Direct clone analysis and selection technology

ABSTRACT

The present invention describes a spatial addressing technique that uses a very high-density micro-pore array for high-throughput screening of biological interactions. The therapeutic, diagnostic, and drug-discovery implications of being able to identify, select and characterize specific protein-protein, protein-DNA and/or protein-carbohydrate interactions from heterogeneous populations of millions (to billions) of cells is discussed. Importantly, this technique possesses the screening and selection capacity of current display-based screening systems (i.e. millions-billions) but with greater efficiency and shorter time.

FIELD OF INVENTION

The invention is related to high throughput assay screening technologyplatforms. The platforms described herein can be used to discover,characterize and select specific interaction pairs from a heterogeneouspopulation of millions or even billions of cells (i.e., for example,bacterial clones). For example, a very high-density micro-pore array isscreened and, after screening, cells are collected from selectedmicro-pores. Optionally, the micro-pore array is reversibly attached toa solid substrate, wherein, after screening, the array is removed fromthe substrate for cell collection from selected micro-pores.

BACKGROUND

There are many technologies used commercially to select and screencompounds from large diverse protein libraries. These technologiesinclude Phage Display, Ribosome Display, Yeast Display and BacterialDisplay, in vitro compartmentalization, microengraving and spatialaddressing. Lin et al., (2002). “Screening and Selection Methods forLarge-Scale Analysis of Protein Function” Angew. Chem. Int. Ed.,41:4402-4425 (2002); Willats et al., (2002) “Phage display:practicalities and prospects” Plant Molecular Biology 50;837-854; andSergeeva et. al., (2006) “Display technologies: Applications for thediscovery of drug and gene delivery agents” Advanced Drug DeliveryReviews 58:1622-1654.

The advent of phage display technology and alternative display systemsallowed antibody screening by linking them to bacterial viruses andprovided recovery of the antibody genes post screening by infection intobacteria. The technology has been widely commercialized. Due mainly tothe costs of these licenses and the competitive nature of the lucrativeantibody-based therapeutic market, alternative display technologies havealso been developed. For example, microengraving microwells may capturesingle cells. Nonetheless, the current techniques lack the necessaryrobustness and selectivity to provide large-scale diagnostic andtherapeutic screening.

A single-cell analysis in quantitative biology has been explored byvarious methods. Levsky et al., Science 297:836-840 2002; Hong et al.,Nat. Biotechnol. 22:435-439 2004; Kurimoto et al., Nucleic Acids Res.34:e42 (2006); Huang et al., Science 315:81-84 (2007); and Newman etal., Nature 441:840-846 (2006). However, these newly emerging methodshave not been fully applied in the biological sciences as of yet. One ofthe reasons for this is the fact that these methods are toosophisticated and integrated to be used without appropriate investmentof time, money, and labor. Thus, there is a strong need to simplify andmake these methods more user-friendly for realization of quantitativebiology at the single-cell level.

What is needed in the art is a better system for direct selection andcharacterization for processing cells and/or interacting biochemicalpairs and selectively isolating each single cell.

SUMMARY

The invention is related to high throughput assay screening technologyplatforms. The platforms described herein can be used to discover,characterize and select specific interaction pairs from a heterogeneouspopulation of millions or even billions of cells (i.e., for example,bacterial clones). For example, a very high-density micro-pore array isscreened and, after screening, cells are collected from selectedmicro-pores. Optionally, the micro-pore array is reversibly attached toa solid substrate, wherein, after screening, the array is removed fromthe substrate for cell collection from selected micro-pores.

In one embodiment, the present invention contemplates a devicecomprising an array of micro-pores, the micro-pore array beingreversibly attached to a solid substrate, wherein at least one bindingpartner is attached to said solid substrate, and wherein the internaldiameter of the micro-pores ranges between approximately 1.0 micrometersand 500 micrometers. Optionally, the micro-pores are not coated with atleast one binding partner. Optionally, said each of said micro-pores hasan internal diameter in the range between approximately 1.0 micrometersand 300 micrometers; optionally between approximately 1.0 micrometersand 100 micrometers; further optionally between approximately 1.0micrometers and 75 micrometers; still further optionally betweenapproximately 1.0 micrometers and 50 micrometers, still furtheroptionally between approximately 5.0 micrometers and 50 micrometers.Optionally, there are approximately 300 to 1,150,000 of saidmicro-pores, per cm² of said array. In one embodiment, the devicefurther comprises a polymeric film, wherein said array is covered bysaid polymeric film, wherein said polymeric film further comprises atleast one hole and wherein said hole is positioned over at least one ofsaid micro-pores. In one embodiment, the device further comprises apressure source configured proximal to said at least one hole. Such apressure source may comprise a pressurized fluid source. Optionally,said micro-pores further comprise at least one biological cell; furtheroptionally, said at least one biological cell is a transformed microbialor mammalian cell that, optionally, secretes a recombinant antibody. Inone embodiment, the micro-pores range between approximately 10micrometers and 1 millimeter long. In one embodiment, the micro-poresrange between approximately 10 micrometers and 1 centimeter long. In oneembodiment, the micro-pores range between approximately 10 micrometersand 10 millimeter long. In one embodiment, the micro-pores range betweenapproximately 10 micrometers and 100 millimeter long. In one embodiment,the micro-pores range between approximately 0.5 millimeter and 1 meterlong. In one embodiment, the micro-pores are approximately 1 millimeterlong. In one embodiment, the micro-pores are approximately 10millimeters long. In one embodiment, the micro-pores are approximately 1centimeter long. In one embodiment, the micro-pores are approximately 1meter long. In one embodiment, each of the micro-pores defines anopening that is approximately 5 micrometers in diameter. In oneembodiment, each of the micro-pores defines an opening that isapproximately 10 micrometers in diameter. In one embodiment, each of themicro-pores defines an opening that is approximately 15 micrometers indiameter. In one embodiment, each of the micro-pores defines an openingthat is approximately 25 micrometers in diameter. In one embodiment,each of the micro-pores defines an opening that is approximately 50micrometers in diameter. In one embodiment, each of the micro-poresdefines an opening that is approximately 100 micrometers in diameter. Inone embodiment, each of the micro-pores defines an opening that isapproximately 300 micrometers in diameter. In one embodiment, each ofthe micro-pores defines an opening that is approximately 500 micrometersin diameter.

In one embodiment, the present invention contemplates a devicecomprising a plurality of longitudinally fused fibers reversibly bondedto a single degassed solid substrate that is gas permeable, wherein thesolid substrate is attached to at least one binding partner. In oneembodiment, the fibers comprise glass capillary fibers. In oneembodiment, the fused capillary fibers are not attached to at least onebinding partner. In one embodiment, the fused capillary fibers areattached to at least one binding partner. In one embodiment, the devicecomprises a micro-pore testbed array. In one embodiment, the fusedcapillary fibers range between approximately 10 micrometers and 1millimeter long. In one embodiment, the fused capillary fibers rangebetween approximately 0.5 millimeter and 1 meter long. In oneembodiment, the fused capillary fibers are approximately 1 millimeterlong. In one embodiment, the fused capillary fibers are approximately 10millimeters long. In one embodiment, the fused capillary fibers areapproximately 1 centimeter long. In one embodiment, the fused capillaryfibers are approximately 1 meter long. In one embodiment, each of thefused capillary fibers range between approximately 5 micrometers and 500micrometers in diameter. In one embodiment, each of the fused capillaryfibers range between approximately 2 micrometers and 500 micrometers indiameter; or between approximately 1.0 micrometers and 500 micrometers,optionally between approximately 1.0 micrometers and 300 micrometers;further optionally between approximately 1.0 micrometers and 100micrometers; further optionally between approximately 1.0 micrometersand 75 micrometers; still further optionally between approximately 1.0micrometers and 50 micrometers, still further optionally, betweenapproximately 5.0 micrometers and 50 micrometers. In one embodiment,each of the fused capillary fibers is approximately 5 micrometers indiameter. In one embodiment, each of the fused capillary fibers isapproximately 10 micrometers in diameter. In one embodiment, each of thefused capillary fibers is approximately 15 micrometers in diameter. Inone embodiment, each of the fused capillary fibers is approximately 25micrometers in diameter. In one embodiment, each of the fused capillaryfibers is approximately 50 micrometers in diameter. In one embodiment,each of the fused capillary fibers is approximately 100 micrometers indiameter. In one embodiment, each of the fused capillary fibers isapproximately 300 micrometers in diameter. In one embodiment, each ofthe fused capillary fibers is approximately 500 micrometers in diameter.In one embodiment, the plurality of fused capillary fibers rangesbetween approximately 300,000 and 5,000,000,000 capillary fibers, eachfiber defining a well. In one embodiment, there are betweenapproximately 300 to 1,150,000 of said fused fibers, per cm² of thearray. In one embodiment, there are between approximately 300 of saidfused fibers, per cm² of the array. In one embodiment, there are betweenapproximately 1,000 to 1,150,000 of said fused fibers, per cm² of thearray. In one embodiment, there are between approximately 10,000 of saidfused fibers, per cm² of the array. In one embodiment, there are betweenapproximately 50,000 of said fused fibers, per cm² of the array. In oneembodiment, there are between approximately 500,000 of said fusedfibers, per cm² of the array. In one embodiment, there are betweenapproximately 1,150,000 of said fused fibers, per cm² of the array. Inone embodiment, the device further comprises at least one biologicalcell in each of the wells. In one embodiment, the plurality of fusedcapillary fibers is approximately 300,000 fused capillary fibers. In oneembodiment, the plurality of fused capillary fibers is approximately500,000 fused capillary fibers. In one embodiment, the plurality offused capillary fibers is approximately 1,000,000 fused capillaryfibers. In one embodiment, the plurality of fused capillary fibers isapproximately 5,000,000 fused capillary fibers. In one embodiment, theplurality of fused capillary fibers is approximately 1,000,000,000 fusedcapillary fibers. In one embodiment, the plurality of fused capillaryfibers is approximately 5,000,000,000 fused capillary fibers. In oneembodiment, the solid substrate comprises silicon. In one embodiment,the solid substrate is polymer. In one embodiment, the gas permeablematerial comprises poly(dimethylsiloxane) (PDMS). In one embodiment, thesolid substrate comprises glass. In one embodiment, the solid substratecomprises quartz. In one embodiment, the solid substrate is degassed andthe solid substrate is a gas permeable material. In another embodiment,the solid substrate is coated with an agent such as vinyl silane oraminopropyltriethoxy silane (APTES), which may be useful to allowattachment of the solid substrate (for example, glass) to the at leastone binding partner.

In one embodiment, the present invention contemplates a devicecomprising a plurality of longitudinally fused fibers and a polymericfilm, wherein the fused fibers are bonded to the polymeric film. In oneembodiment, the fused fibers comprise fused glass capillary fibers.

In one embodiment, the polymeric film further comprises at least onehole, wherein the hole is positioned over at least one of the capillaryfibers. In one embodiment, the device further comprises a pressuresource comprising a nozzle, wherein the nozzle is configured to fitwithin the circumference of the at least one hole. In one embodiment,the capillary fiber further comprises at least one biological cell. Inone embodiment, the at least one biological cell is a transformedbiological cell. In one embodiment, the biological cell comprises amicrobial, fungal, mammalian, insect or animal cell. In one embodiment,the microbial cell comprises a bacterial cell. In one embodiment, thebacterial cell comprises an E. coli cell. In one embodiment, the cellcomprises a fungal cell. In one embodiment, the cell comprises amammalian cell. In one embodiment, the microbial cell is a transformedmicrobial cell. In one embodiment, the cell comprises a transformedfungal cell. In one embodiment, the cell comprises a transformedmammalian cell. In one embodiment, the transformed microbial cellsecretes a recombinant antibody. In one embodiment, the transformedmicrobial cell secretes a recombinant protein and/or peptide. In oneembodiment, the biological cell is an animal cell In one embodiment, theanimal cell comprises a rare biochemical compound. In one embodiment,the rare biochemical compound is selected from the group comprising aprotein, a peptide, a hormone, a nucleic acid, a carbohydrate.

In one embodiment, the fused capillary fibers range betweenapproximately 0.5 millimeter and 1 meter long. In one embodiment, eachof the fused capillary fibers range between approximately 5 micrometersand 500 micrometers in diameter. In one embodiment, the hole rangesbetween approximately 5 micrometers and 500 micrometers in diameter. Inone embodiment, the plurality of fused capillary fibers ranges betweenapproximately 300,000 and 5,000,000,000 capillary fibers. In oneembodiment, the polymeric film is selected from the group including butnot limited to polylactide, polygalactide, polypropylene, polybutylene,polycaprone, polyester and any combination thereof.

In one embodiment, the present invention contemplates a method foridentifying a sub-population of cells from a heterologous population ofbiological cells, the method comprising: a) providing: i) an array ofmicro-pores, wherein the internal diameter of micro-pores ranges betweenapproximately 1.0 micrometers and 500 micrometers; ii) said heterologouspopulation of cells; iii) at least one binding partner; b) contactingsaid array with said heterologous population of cells and said at leastone binding partner such that a sub-population comprising at least oneof said biological cells settles into at least one of said micro-poresof said array; c) incubating said array under conditions to promote thesecretion of molecules from said biological cells; and d) detectingdesired secreted molecules in at least one of said micro-pores of saidarray, thereby identifying said sub-population of cells. In oneembodiment, the providing step comprises providing an array ofmicro-pores not being coated with at least one binding partner. In oneembodiment, the detecting step comprises detecting the desired secretedmolecules in association with at least one binding partner.

In one embodiment, the present invention contemplates a methodcomprising, a) providing: i) a degassed solid substrate comprising atleast one binding partner, wherein the solid substrate is gas permeableand is attached to a plurality of longitudinally fused fibers therebycreating a micro-pore testbed array; ii) a plurality of biological cells(the cells being in solution or suspension); iii) optionally a polymericfilm comprising at least one hole, such that the film is configured tobond to the fused capillary fibers; and b) contacting the micro-poretestbed array with the solution such that at least one of the pluralityof biological cells settles into at least one of the fused capillaryfibers; c) optionally bonding the polymeric film to the fused capillaryfibers such that the hole is positioned over at least one of thecapillary fibers; d) removing the fused capillary fiber-polymeric filmarray from the solid substrate; and e) collecting the at least onebiological cell from the at least one fused capillary fiber wherein,optionally, the nozzle of the pressure source is placed over the atleast one capillary fiber. In one embodiment, the solid substrate isreversibly attached to the plurality of longitudinally fused fibers. Inone embodiment, the method further comprises providing a pressure sourceconfigured proximal to said at least one hole. In one embodiment, thepressure source comprises a nozzle. In one embodiment, the methodfurther comprises collecting a sub-population, such as the plurality ofbiological cells from said at least one fused fiber. In one embodiment,the fibers comprise glass capillary fibers. In one embodiment, the fusedcapillary fibers do not comprise at least one binding partner. In oneembodiment, the plurality of biological cells release and/or secrete atleast one biological compound having affinity for the at least onebinding partner. In one embodiment, the at least one binding partner maybe selected from the group comprising antigens, antibodies, proteins,peptides, nucleic acids, deoxyribonucleic acids, ribonucleic acids,lipids, and/or carbohydrates. In one embodiment, the method furthercomprising binding the at least one binding partner to the at least onebiological compound, wherein a binding partner/biological compoundcomplex is formed on the solid substrate. In one embodiment, the methodfurther comprises detecting the binding partner/biological compoundcomplex. In one embodiment, the detecting comprises a labeled reagenthaving affinity for the binding/partner/biological compound complex. Inone embodiment, the collecting comprises cultivating the at least onebiological cell.

In one embodiment, the present invention contemplates a methodcomprising: a) providing: i) a plurality of longitudinally fused fibersreversibly attached to a solid support, each fiber defining a well, thesolid surface comprising at least one type of binding partner andserving as the bottom of the well, the wells collectively comprising anarray; ii) a heterologous population of biological cells (in solution orsuspension); b) contacting said array with said solution such that atleast one of said cells settles into at least one of said wells of saidarray; c) incubating said array under conditions to promote thesecretion of molecules from said cells; d) removing said plurality offused fibers from said solid support; and e) detecting whether secretedmolecules bound said binding partner on said solid support. In oneembodiment, the fibers comprise glass capillaries.

In one embodiment, the method of the present invention contemplates theconcentration of the suspension of heterologous population of cells andthe dimensions of the array are arranged such that 1-1000 cells,optionally, 1-500 cells, further optionally, 1-100 cells, still furtheroptionally 1-10 cells, still further optionally, 1-5 cells, aredistributed into at least one of said micro-pores of the array.

In one embodiment, the present invention contemplates a kit comprising:a first container comprising an array of micro-pores, wherein theinternal diameter of micro-pores range between approximately 1.0micrometers and 500 micrometers; and a second container comprising atleast one binding partner. In one embodiment the second containercomprises a solid substrate comprising said at least one bindingpartner, the solid substrate being capable of reversible attachment tothe array of micro-pores.

In one embodiment, the present invention contemplates a kit comprising afirst container comprising a micro-pore array comprising a plurality offused capillary fibers that are not coated with at least one bindingpartner. In one embodiment, the kit further comprises a second containercomprising a degassed solid substrate comprising at least one bindingpartner. In one embodiment, the kit further comprises a third containercomprising a plurality of labeled reagents capable of detecting avariety of binding partner-biological compound complexes. In oneembodiment, the kit further comprises a fourth container comprising asolution comprising a biological cell comprising a recombinant protein.In one embodiment, the kit further comprises instructional materialscontaining directions (i.e., protocols) providing for the use of themicro-pore array in the detection of various biological compounds thatare secreted from a biological cell.

In one embodiment, the present invention contemplates use of an array ofmicro-pores for identifying a selected sub-population of cells from aheterologous population of biological cells, the use comprising: a)providing: i) an array of micro-pores, the diameter of micro-poresranging between approximately 2.0 micrometers and 500 micrometers indiameter; ii) a heterologous population of cells; b) contacting saidarray with said solution such that the sub-population settles into atleast one of said micro-pores of said array; c) incubating said arrayunder conditions to promote the secretion of molecules from said cells;and d) detecting desired secreted molecules in at least one of saidmicro-pores of said array. In one embodiment, the array is not coatedwith at least one binding partner. In one embodiment, the concentrationof the suspension of heterologous population of cells and the dimensionsof the array are arranged such that 1-1000 cells, optionally, 1-500cells, further optionally, 1-100 cells, still further optionally 1-10cells, still further optionally, 1-5 cells, are distributed into atleast one of said micro-pores of the array.

DEFINITIONS

The terms “binding partner”, “ligand” or “receptor” as used herein, maybe any of a large number of different molecules, or aggregates, and theterms are used interchangeably. Each binding partner may be immobilizedon a solid substrate and binds to an analyte being detected.Alternatively, each binding partner may not be immobilized on either thesolid substrate or on the micro-pores of the array. Proteins,polypeptides, peptides, nucleic acids (nucleotides, oligonucleotides andpolynucleotides), antibodies, ligands, saccharides, polysaccharides,receptors, antibiotics, test compounds (particularly those produced bycombinatorial chemistry), may each be a binding partner. Onenon-limiting example of binding partners are antibodies and antigens.

The term “biological cell” as used herein, refers to any cell from anorganism, including, but not limited to, microbial, fungal (for example,yeast) or animal, such as mammalian or insect, cells.

The terms “biological compound” or “analyte” as used herein, refers toany compound released (i.e., for example, secreted) by a biologicalcell. Such a compound or analyte may be an amino acid sequence, anucleic acid sequence, a hormone or any other biologically synthesizedmolecule. The biological compounds or analyte may attach to a bindingpartner, wherein they are detected as a binding partner/compound orbinding partner/analyte complex.

The term “longitudinally fused fiber” as used herein, refers to at leasttwo fibers that are attached along their respective longitudinal axissuch that they become a single unit. Such attachment may be facilitatedby exposure to heat, chemicals, or adhesives. Such fibers are generallyhollow and may be comprised of glass capillary fibers.

The term “reversibly bonded” or ‘reversibly attached” as used herein,refers to any attachment between to device components that provides awatertight seal while bonded, but may be separated without the use ofchemicals or heat. For example, the device components may be separatedby hand.

The term “degassed solid substrate” as used herein, refers to anymaterial capable of supporting the reversible bonding of a micro-porearray, wherein the material has a decreased content of dissolved gases.For example, a degassed solid substrate may comprise PDMS. However, itwill be appreciated that solid substrates that are not degassed are alsocapable of supporting the reversible bonding of a micro-pore array.

The term “micro-pore testbed array” as used herein, refers to anyassembly comprising a micro-pore array reversibly bonded to a solidsubstrate.

The term “cultivating” or “culturing” as used herein, refers to anymethod wherein a cell or plurality of cells in incubated in a mediumthat supports cell proliferation such that the number of living cellsincrease.

The term “polymeric film” as used herein, refers to any sheet ofmaterial capable of forming a seal with the open ends of a micro-poremicroarray and susceptible to penetration by a low energy laser, therebycreating a hole (i.e., for example, between 5-500 μm in diameter).

The term “rare biochemical compound” as used herein, refers to anybiochemical compound that is produced in less than between approximately20%-0.001% of native biological cells.

The term “pressure source” as used herein, refers to any device capableof generating a mechanical, liquid or gaseous stream at a variety ofpressures, both positive and negative. Preferably, the diameter of theliquid or gaseous stream may be varied by using an adjustable nozzle.For example, one pressure source may comprise an air pressure source.The pressure source may be configured proximal to at least one of thefused fibers, wherein the pressure source contents may be expelledthereby removing the contents of the fused fibers or micro-pores.Optionally, a fused fiber or micro-pore may be covered with a polymericfilm comprising a hole in-line with the fused fibers or micro-pores,wherein the hole may have a diameter greater than, equal to or smallerthan the fused fiber diameter or the inner diameter of the micro-pore.

The term “secrete” as used herein, refers to any release of a biologicalcompound or analyte from a biological cell in to the surrounding medium.Such secretion may be the result of active transport, passive diffusionor cell lysis.

The term “bind” or “attach” as used herein, includes any physicalattachment or close association, which may be permanent or temporary.Generally, an interaction of hydrogen bonding, hydrophobic forces, vander Waals forces, covalent and ionic bonding etc., facilitates physicalattachment between the molecule of interest and the analyte beingmeasured. The “binding” interaction may be brief as in the situationwhere binding causes a chemical reaction to occur. That is typical whenthe binding component is an enzyme and the analyte is a substrate forthe enzyme. Reactions resulting from contact between the binding agentand the analyte are also within the definition of binding for thepurposes of the present invention.

The term “fiber” as used herein, includes both filaments and hollowcapillary structures. Pluralities, typically a large number, of fibersare bound (i.e., for example, fused) adjacent to each other in ribbonsor bundles to form a “block.” A fiber block may constitute a portion ofthe actual bundle being used. A cross-section of the fibers may be ofany shape, such as round, triangular, square, rectangular or polygonal.The fibers may be of material such as glass, metal, ceramic or plastic.

The term “sintering” as used herein, refers to the fusion of thesurfaces of the fibers without actually melting the whole fiber.Sintering may be chemical or thermal and may even involve aself-adhesive component that may be activatable.

The terms “arrays” and “microarrays” are used somewhat interchangeablydiffering only in general size. The instant invention involves the samemethods for making and using either. Each array typically contains manycells (typically 100-1,000,000+) wherein each cell is at a knownlocation and contains a specific component of interest. Each arraytherefore contains numerous different components of interest.

In a related aspect, “device” is used to describe both arrays andmicroarrays, where the array or microarray may comprise other definedcomponents including surfaces and points of contact between reagents.

Further, “substrate” is also a term used to describe surfaces as well assolid phases which may comprise the array, microarray or device. In somecases, the substrate is solid and may comprise PDMS.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatconsist of amino acid residues joined by peptide bonds, contain theelements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general,a protein comprises amino acids having an order of magnitude within thehundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

The term, “purified” or “isolated”, as used herein, may refer to apeptide composition that has been subjected to treatment (i.e., forexample, fractionation) to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the composition(i.e., for example, weight/weight and/or weight/volume). The term“purified to homogeneity” is used to include compositions that have beenpurified to ‘apparent homogeneity” such that there is single proteinspecies (i.e., for example, based upon SDS-PAGE or HPLC analysis). Apurified composition is not intended to mean that some trace impuritiesmay remain.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and more preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

“Nucleic acid sequence” and “nucleotide sequence” as used herein referto an oligonucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.

The term “an isolated nucleic acid”, as used herein, refers to anynucleic acid molecule that has been removed from its natural state(e.g., removed from a cell and is, in a preferred embodiment, free ofother genomic nucleic acid).

The terms “amino acid sequence” and “polypeptide sequence” as usedherein, are interchangeable and refer to a sequence of amino acids.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

The term “antibody” refers to immunoglobulin evoked in a host by animmunogen (antigen) or the expression product of cloned human or animalimmunoglobulin genes by semi-synthetic (modified post cloning by PCR) orfully synthetic techniques (made in vitro and diversified by PCR). It isdesired that the antibody demonstrates specificity to epitopes containedin the immunogen. An antibody may also comprise antibody fragments suchas single chain fragment variable (scFv), Fragment binding (Fab) and anyother format of recombinant antibody fragment. The term “polyclonalantibody” refers to immunoglobulin produced from more than a singleclone of plasma cells; in contrast “monoclonal antibody” refers toimmunoglobulin produced from a single clone of plasma cells.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., for example, an antigenic determinant orepitope) on a protein; in other words an antibody is recognizing andbinding to a specific protein structure rather than to proteins ingeneral. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabelled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

The term “small organic molecule” as used herein, refers to any moleculeof a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size from approximately 10 Da up to about 5000 Da, more preferably upto 2000 Da, and most preferably up to about 1000 Da.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables). For example, a pulmonary sample may becollected by bronchoalveolar lavage (BAL), which comprises fluid andcells derived from lung tissues. A biological sample may comprise acell, tissue extract, body fluid, chromosomes or extrachromosomalelements isolated from a cell, genomic DNA (in solution or bound to asolid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like.

The term “biologically active” refers to any molecule having structural,regulatory or biochemical functions. For example, biological activitymay be determined, for example, by restoration of wild-type growth incells lacking protein activity. Cells lacking protein activity may beproduced by many methods (i.e., for example, point mutation andframe-shift mutation). Complementation is achieved by transfecting cellsthat lack protein activity with an expression vector which expresses theprotein, a derivative thereof, or a portion thereof.

The term “immunologically active” defines the capability of a natural,recombinant or synthetic peptide, or any oligopeptide thereof, to inducea specific immune response in appropriate animals or cells and/or tobind with specific antibodies.

The term “antigenic determinant” as used herein refers to that portionof a molecule that is recognized by a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms “immunogen,” “antigen,” “immunogenic” and “antigenic” refer toany substance capable of generating antibodies when introduced into ananimal. By definition, an immunogen must contain at least one epitope(the specific biochemical unit capable of causing an immune response),and generally contains many more. Proteins are most frequently used asimmunogens, but lipid and nucleic acid moieties complexed with proteinsmay also act as immunogens. The latter complexes are often useful whensmaller molecules with few epitopes do not stimulate a satisfactoryimmune response by themselves.

As used herein, the terms “complementary” or “complementarity” are usedin reference to “polynucleotides” and “oligonucleotides” (which areinterchangeable terms that refer to a sequence of nucleotides) relatedby the base-pairing rules. For example, the sequence “C-A-G-T,” iscomplementary to the sequence “G-T-C-A.” Complementarity can be“partial” or “total.” “Partial” complementarity is where one or morenucleic acid bases is not matched according to the base pairing rules.“Total” or “complete” complementarity between nucleic acids is whereeach and every nucleic acid base is matched with another base under thebase pairing rules. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods thatdepend upon binding between nucleic acids.

The term “transfection” or “transfected” refers to the introduction offoreign DNA into a cell.

As used herein, the terms “nucleic acid molecule encoding”, “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

The term “label” or “detectable label” are used herein, to refer to anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Such labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein,Texas red, rhodamine, green fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴ _(C), or, ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and calorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include, but are not limited to,U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241 (all herein incorporated by reference). Thelabels contemplated in the present invention may be detected by manymethods. For example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting, the reaction product produced by the action of the enzyme onthe substrate, and calorimetric labels are detected by simplyvisualizing the colored label.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates one embodiment of a very high-density micro-porearray, having 5 μm pores.

FIG. 2 illustrates one embodiment of making a degassed glass micro-poretestbed array, the testbed array comprising a micro-pore arrayreversibly attached to a solid substrate.

FIG. 3 illustrates one embodiment for loading the micro-pores of a glassmicro-pore testbed array.

FIGS. 4A and 4B illustrate one embodiment of a biological recognitionassay performed within each micro-pore. For example, the recognitionassay may comprise an antigen-antibody recognition assay.

FIG. 5 illustrates one embodiment of a method to select and isolate asub-population (for example, a cell) from a very high-density micro-porearray.

FIG. 6 illustrates one embodiment of a method for scanning andaddressing (locating) the positive binding sites (i.e., high intensityspots), thereby allowing subsequent isolating and extraction ofcell-specific DNA.

FIGS. 7A-7C present a representative glass micro-pore array. Scale barsare 20 μm. FIG. 7A presents a glass micro-pore array placed onto adegassed PDMS solid substrate (red box enlarged in FIG. 7B). FIG. 7Bpresents a microscopic image of the surface of the micro-pore array.FIG. 7C presents heterogeneous dye staining from different pores ontothe PDMS surface.

FIG. 8 presents exemplary data showing a phage-biotin labeledanti-phage—FITC streptavidin assay that reveals a signal that isdependent on the antibody concentration.

FIG. 8A presents exemplary data showing immunoassay performance on themicropore filter array.

FIGS. 9A and 9B present exemplary data of E. coil expressing GFP proteinloaded at different densities (FIG. 9A—neat; FIG. 9B—1/100 of neat)inside the micro-pore test bed array after incubation for 1.5 hrs. Allpores are ˜10 μm diameter.

FIG. 10 presents exemplary real time fluorescence data from cellsinduced to produce GFP in a micro-pore testbed array (open diamonds).Negative control, heat inactivated cells demonstrated no production ofGFP (open squares).

FIG. 10B presents exemplary data showing visualisation of fluorescentimages from the positive induced E. coli (top four images) and negativeheat inactivated E. coli (bottom four images) cells in the array. Thefluorescent intensity of each area taken every 5 minutes is representedin FIG. 10.

FIG. 11 presents exemplary data of antibody secretion by IPTG inductionin E. coli clones grown on an ELISA plate demonstrating improvedspecificity and reduced background in comparison to phage displaytechnology.

FIG. 12 presents exemplary data of cell growth and GFP expression inmicrotitre plates with varying cell loading densities.

FIG. 13 shows capture and growth of E. coli cells on agar plates afterrecovery from the micro-array testbed. FIG. 13A shows a 1/10 celldilution: No single colonies were observed as the concentration of thecells removed from the array was very high. The cells grew well on theagar plates. FIG. 13B shows a 1/100 cell dilution: Single colonies wereobserved but were still hard to differentiate as the concentration ofthe cells removed from the array was very high. The cells grew well onthe agar plates. FIG. 13C shows a 1/1000 cell dilution: A single colonywas observed indicating a low concentration of viable cells in the area.Note: the procedure used to the remove the cells is crude and is notfully yet optimised. Experimental conclusions: The experimentsuccessfully showed that the cells can grow and live in the array andcan be recovered on agar plates for further characterisation. Due to thepore structure of the array of the present invention, the cells could besimply removed by air pressure (like blowing liquid from a straw) hasbeen shown on a microscale for single pore analysis.

FIG. 14 presents a generalized overview of conventional high throughputprocesses of identifying interaction pairs (pairs of binding partners)in parallel.

FIG. 15 presents one embodiment for constructing a fused block ofcapillary fibers or a micro-pore array.

FIG. 16 present several commercially available glass capillary fibercompositions or micro-pore arrays, useful in the present invention (Cat#'s: J5022-01, J5022-09, J5022-11, J5022-16, J5022-19 J5022-21;Hamamatsu, Japan).

FIG. 17 presents one embodiment of a pressure-based cell selectionmethod. A plastic sheet printed with black laser printer ink was laseredto create a 100 μm diameter laser hole and was placed over the glassarray which had been filled with food colorant.

FIG. 17A presents another embodiment of a pressure-based cell selectionmethod. A plastic sheet printed with black laser printer ink was laseredto create a 40 82 m diameter laser hole and was placed over the glassarray which had been filled with food colorant.

FIG. 18 presents exemplary data showing the removal of food colorantafter exposure to air pressure through an ˜100 μm hole in a plastic film(plastic transparent sheet with laser printed black area (to absorb theenergy from the laser and ablate)). The air pressure removed thecolorant almost completely from the pores in a 300 μm area around thelaser hole (white area).

FIG. 18A presents exemplary data showing the removal of food colorantafter exposure to air pressure through an ˜40 μm hole in Scotch tape .The air pressure removed the colorant almost completely from the poresin a 40 μm area around the laser hole (white area).

FIG. 18B presents exemplary data showing single pore recovery of viableE. coli cells from a 40 μm diameter micro-pore array.

FIGS. 19A, 19B and 19C illustrate some of the primary advantages of thedirect clone analysis and selection technique described herein, ascompared to conventional ribosomal, phage display, and microengravingscreening techniques.

FIG. 20 demonstrates that surface tension actually prevents a liquiddrop (suspension of heterologous cells, for example) from entering thecentral micro-pores.

FIG. 21 demonstrates that, when the drop is spread evenly over themicro-pore array surface, the surface tension is removed and allmicro-pores are filled evenly.

FIGS. 22A, 22B and 22C present exemplary data showing the elimination ofa halo ring effect following detection of the appropriate analyte whenthe solution drop is spread evenly across the top of the micro-porearray.

FIG. 23 demonstrates that specific B cell antibody response can bedetected when cultured in the array and individual positive pores can bevisualized.

FIG. 24 illustrates that specific B cell antibody response can bedetected when cultured in the array

FIG. 25 demonstrates that specific recombinant antibody response can bedetected when cultured in the array.

FIG. 26 demonstrates non-degassed driven loading and patterning of themicropore array. This Figure is to show that even patterning can beachieved without leckage to air blockage, without degas driven flow.

FIG. 26B demonstrates a comparison of immunoassay performance fromdegassed and non-degassed driven loading of the micropore array. ThisFigure shows that the immunoassay performance in the array is notaffected by whether the solid substrate (such as PDMS), if present, isdegassed or non-degassed.

FIG. 27 demonstrates enhanced surface coating with APTES coated PDMS.

FIGS. 28A and 28B demonstrate that cells residing in target pores may beharvested. FIG. 28A illustrates one embodiment of a pore retrieval setupconsisting of an array holder mounted on to a high precision XY stage, astationary laser nozzle and nitrogen supply and a 384 well plate placedonto a second high precision XY stage.

FIG. 29 presents exemplary data demonstrating of reassembly of GFP ‘invivo’ in liquid media. Cells have been pelleted by centrifugation at4000 rpm for 10 mins and the supernatant removed.

FIG. 30 presents exemplary data showing a graphical representation ofthe fluorescent intensity values obtained from microarray pore arraywith E. coli cultures harbouring split GFP fragments using a fluorescentmicroscope.

FIG. 31 presents exemplary data showing an emission scan of the DNA-FRETdue to binding of complimentary 21 bp DNA probes labelled with Cy5(donor) and Cy5.5 (acceptor).

FIG. 32 presents exemplary data showing mean and maximum emissionintensities of the DNA-FRET due to binding of complimentary 21 bp DNAprobes labelled with Cy5 (donor) and Cy5.5 (acceptor) in a 40 μmdiameter micropore array.

DETAILED DESCRIPTION

The invention is related to high throughput assay screening technologyplatforms. The platforms described herein can be used to discover,characterize and select specific interaction pairs from a heterogeneouspopulation of millions or even billions of cells (i.e., for example,bacterial clones). For example, a very high-density micro-pore array isscreened and, after screening, cells are collected from selectedmicro-pores. Optionally, the micro-pore array is reversibly attached toa solid substrate, wherein after screening, the array is removed fromthe substrate for cell collection from selected micro-pores.

Using conventional technologies, biological libraries may be screenedfor components including but not limited to, antibodies, proteins,peptides, nucleic acids, deoxyribonucleic acids, and/or ribonucleicacids. These systems have a number of disadvantages, including the needto enrich for desired clones via repeat selection steps (including, forexample “panning”) that inherently result in the loss of potentialbinding candidates. It is also difficult to establish the precise originof a positive signal using conventional technologies since they obtainmixed signals from heterogeneous populations that cannot be convoluted.Generally these techniques involve selection processes utilizingbacteriophages, ribosomes and specific cells, most of which areperformed in vitro. Improvements in library screening have introducedthe concept of spatial addressing in order to maintain identity of thescreened components during the selection process. Such addressing can bebased upon techniques including robotics, enzyme-linked immunosorbentassays, or cell-based assays. While spatial addressing can, for example,identify specific cellular clones to generate master stocks, theselimitations do not facilitate high throughput screening techniques toselectively isolate and purify the identified clones for rapidapplication to disease diagnostics and therapeutics. Anotherdisadvantage of the present screening assays are that they are usuallylimited to a cell number between approximately 50K -100K. In oneembodiment, the invention provides a simple and direct technique foranalyzing billions of antibody (or desired protein) secreting cellswithout the need for their display on viruses (phage display), ribosomes(ribosomal display) or cells (mammalian, bacterial or yeast display). Inone embodiment, the invention provides a simple and direct technique fordirectly analyzing billions of antibody (or desired protein) secretingcells.

In one embodiment, the present invention contemplates methods thatimprove upon the above-mentioned selection processes by a techniquecomprising direct clone analysis and selection. In one embodiment, thedirect clone analysis method utilizes native cells (i.e., not limited tocell culture stocks) that are cultivated in a micro-pore array, whereinthe micro-pore array is, optionally, not coated with any biologicalligands (i.e., for example, binding partners). In one embodiment, thenative cells comprise fresh tissue cells. In one embodiment, the nativecells comprise microbial cells. In one embodiment, the microbial cellsare transformed with at least one recombinant protein. Further, thedirect clone selection method analyzes all samples (i.e., for example,millions and/or billions) in parallel.

In one embodiment, the present invention contemplates a method forselecting billions of antibody producing biological cell clones using amicro-pore array (for example, aaporous glass array) (optionally,uncoated) that is, optionally, reversibly bonded to a solid substrate(i.e., for example, PDMS). In one embodiment, the micro-pores are filledwith a solution (i.e., for example, a culture media) comprising thebiological cell clones harboring antibody (or any protein of interest)genes. In another embodiment, the micro-pores are filled with a solution(i.e., for example, a culture media) comprising the biological cellclones harboring antibody (or any protein of interest) genes by degassdriven forces. In one embodiment, the cells grow and express antibodiesinto the media, which can react and bind with a binding partner that maynot be immobilized on the solid substrate or the array or,alternatively, with antigen immobilized onto a lower PDMS surface. Whenthe device comprises a micro-pore array and a solid substrate, althoughit is not necessary to understand the mechanism of an invention, it isbelieved that, upon removal of the porous micro-pore array from thesolid substrate, an antigen-antibody complex can be detected by addingfluorescent reagents to the solid substrate. It is further believed thatfluorescent spots on the solid substrate giving a high signal maycorrespond to micro-pores containing cells secreting a specific antibodyand/or protein. When the device comprises a micro-pore array and nosolid substrate, although it is not necessary to understand themechanism of an invention, it is believed that an antigen-antibodycomplex can be detected. In one embodiment, the method further comprisesrecovering the biological cells from the micro-pore array. In oneembodiment, the biological cells comprise antibody secreting cells. Inone embodiment, the biological cells comprise cells secreting afluorescent protein. In one embodiment, the biological cells comprisecells secreting a fluorescent protein fused to a non-fluorescentprotein. In one embodiment, the biological cells secreting a fluorescentprotein fused to a non-fluorescent protein is detected directly on asolid substrate that does not have an antigen or antibody immobilizedthereon.

The present invention is not in any way limited and may be used toisolate any types of biological cells, including, but not limited to,cell lines that express or produce proteins, carbohydrates, enzymes,peptides, hormones, receptors; other cell lines that produce antibodies;genetically engineered cells; and activated cells. Moreover, the presentinvention may be used to screen for a variety of biological activitiesincluding, but not limited to, the expression of surface receptorproteins, enzyme production, and peptide production. Furthermore, thepresent invention may be used to screen a variety of test agents todetermine the effect of the test agents on the desired biologicalactivity. Other types of cells desired to be isolated and screened,other types of biological activity desired to be detected, and specifictest agents to be screened will be readily appreciated by one of skillin the art.

Some embodiments of the present invention provide an ability to generateand compare the activity of billions of biological cell variants.Although it is not necessary to understand the mechanism of aninvention, it is believed that these embodiments not only allows theengineering of proteins and cells with new properties, but also providesa powerful new tool for understanding protein structure and function.

I. Conventional Screening Techniques

The need to isolate small numbers of specific cells from backgroundpopulations is ubiquitous, with applications in pathology, clinicaldiagnosis, cloning, and cell biology research. In the context of cellbiology experiments, sorting can be a way to select a desired startingpopulation of cells of known characteristics, or can be a tool toanalyze the results of an experiment and isolate particularlyinteresting cells for further investigation. Eisenstein M., Nature441:1179 (2006).

Selection of hybridomas has involved screening antibodies produced bylarge numbers of cells and retrieving those cells that produceantibodies of desired specificity. Cloning by a limiting serial-dilutionrequires deposition of cells into wells of a microtiter plate (usually96 or 384 wells) such that individual cells are deposited in roughly oneout of three wells. After 5-10 d in culture, the supernatants from eachwell are tested, and the process of dilution is repeated untilmonoclonality is achieved. Two factors determine the time required toisolate a single monoclonal hybridoma by the method of Fuller et al.,In: Current Protocols in Molecular Biology (eds. Ausubel, F. M. et al.)11.8.1-11.8.2, (John Wiley & Sons, Inc., New York, 2003); and Yokoyama,W. M., In: Current Protocols in Immunology (eds. Coligan, J. E.,Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. & Strober, W.)2.5.1-2.5.17, (John Wiley & Sons, Inc., New York, 1995). First, thesensitivity of the assay used to detect antibodies sets the frequencyfor testing; for example, sufficient concentrations of antibodies fordetection by enzyme-linked immunosorbent assays (ELISA) are achieved7-10 d after seeding individual cells into wells. Second, the totalnumber of manipulations limits the number of clones that can be screenedefficiently in any single round of selection (10-100 plates/screen).

One alternative for sorting cells into microtiter plates at limitingdilutions includes picking clones from semi-solid medium. Davis et al.,“A simple, single-step technique for selecting and cloning hybridomasfor the production of monoclonal antibodies” J. Immunol. Methods50:161-171 (1982); and Rueda et al., “Cloning of myelomas and hybridomasin fibrin clots” J. Immunol. Methods 114:213-217 (1988). Anotheralternative for sorting cells includes fluorescence-activated cellsorting (FACS). Herzenberg et al., “The history and future of thefluorescence activated cell sorter and flow cytometry: a view fromStanford” Clin. Chem. 48: 1819-1827 (2002); and Carroll et al., “Theselection of high-producing cell lines using flow cytometry and cellsorting” Expert Opin. Biol. Ther. 4:1821-1829 (2004). Cells plated inhydrogels are challenged to survive and grow slowly, whereas for FACS,the correlation is not straightforward between cells that stain positiveand those that actually secrete. Both methods have improved theefficiency of screening by serial dilution.

A soft lithographic technique has been reported that supports amicroengraving technique that uses microwell arrays. These microwellsmay contain individual cells to identify a corresponding array ofmolecules (including antigen-specific antibody) secreted by each cell.Love et al. (2006) “A Microengraving Method For Rapid Selection OfSingle Cells Producing Antigen-Specific Antibodies” Nature Biotechnology24(6): 703-707. Microengraving arrays are limited to processingapproximately 100,000 individual cells in a system that identifies,recovers, and clonally expands antigen-specific antibody producingcells. Microengraving arrays are fabricated by a combination ofphotolithography and replica molding of monolithic slabs of PDMS formingwells that are either 50 μm or 100 μm in diameter and depth. These wellsare then separated by a distance equal to the well diameter (i.e., 50 μmor 100 μm, respectively). Each PDMS slab coated with bovine serumalbumin to reduce non-specific binding of the cells to the substratesurface. After depositing 0.5 ml of a cell suspension (1×10⁵-5×10⁵cells/ml) onto the PDMS surface approximately 1-3 cells may settle into50-75% of the wells and are cultivated for approximately 1 week. A glassslide coated with a specific antigen and/or anti-immunoglobulin antibodypermits the detection of antibody secreted by individual cells. The PDMSmicroengraved array is then sealed against the coated glass slide,inverted, and the cells are incubated for approximately 2-4 hours, suchthat the secreted antibodies bind to the coated glass slide. Followingthe incubation, the glass slide is removed from the PDMS array, and thebinding pairs on the glass slide are detected, whereas the cells withinthe PDMS array are further cultivated. The pattern of detected bindingpairs identify the microwells containing the cells of interest. Thesecells of interest are collected from the microwells by amicromanipulator system (IM-9A, Narishige) fitted with hand-drawncapillaries (GC-1). To withdraw the contents of a well, the array ofmicrowells was positioned on a microscope under a layer of medium (˜1ml), and a capillary with an outer diameter of 100 μm (inner diameter˜50 μm) was positioned directly over the top of an appropriate well. Asmall volume (˜1-5 μl) was withdrawn with the affixed syringe until thecells were removed from the well successfully. The tip was thentransferred into a well of a 96-well plate containing 200 μl medium (10%hybridoma cloning factor) and the cell(s) expelled into the volume. Bothextraction from the microwell and deposition of the cells into anothercontainer (96-well plate) were monitored visually to ensure the transferof the cells into and out of the tip.

One of the major challenges for performing cell based screening is theisolation of small populations of cells in a manner that allows forsubsequent screening procedures. Traditional devices and methods ofisolating cells do not adequately provide for the isolation of smallpopulations of cells without performing steps that potentially modifycellular function or activity. Isolation of cells is not only importantin screening, but also in processes that involve the monitoring,measuring, and/or use of the output of cellular activity or function(e.g. antibody production) for small populations of cells. For example,once a hybridoma fusion is performed and the cells are plated, there areseveral issues that have to be addressed. First, the cells will grow atdifferent rates, thus the point at which one must perform the assay forantibody production to assess positive pools of cells can vary and mayrequire more than one assay point on the same pool of cells. During thisprocess, the rapidly growing cells need to be passaged in order topromote viability and to prevent loss of potentially positive clones.The next step is to perform limiting dilution with the goal of achievingclonal populations. Successive rounds of this process may be required toachieve clonal or near clonal populations. A microfluidic deliverydevice has been reported for isolating and screening a small populationof cells (or individual cells) for biological activity—includingspecific antibody producing hybridomas—with minimal cell manipulation.Wang et al. “Cell Isolation And Screening Device And Method Of UsingSame,” U.S. Pat. No. 7,169,577. The microfluidic delivery deviceincludes microfluidic channels that deliver cells to isolation regionsboth of which are manufactured by photolithography. As few as one tofive cells may be delivered to each isolation region. These isolationregions contain bioaffinity regions containing ligands that bindspecific types of cells to the substrate surface. Once bound to thedevice, the cells may undergo proliferation and then be transferred to amicroarray well. A detecting device may then be inserted into themicroarray well to bind to an antibody that is secreted by the cell, orthe bottom of the microwell is coated with a binding ligand. This methoddoes not contemplate recovering the specific cells for future use oncethe secreted compound has been detected and identified.

Various procedures for obtaining fully human antibodies have beendeveloped, including phage display libraries of artificial antibodyfragments of human origin. Boder et al., “Yeast surface display forscreening combinatorial polypeptide libraries” Nat. Biotechnol.15:553-557 (1997); McCafferty et al., “Phage antibodies: filamentousphage displaying antibody variable domains” Nature 348:552-554 (1990);Winter et al., “Making antibodies by phage display technology” Annu.Rev. Immunol. 12:433-455 (1994). Other methods select antibodies thatare naturally produced in humans. Attempts have been made to establishhybridomas producing human mAbs or Epstein-Barr virus immortalized humanASCs. Kozbor et al., “Requirements for the establishment of high-titeredhuman monoclonal antibodies against tetanus toxoid using theEpstein-Barr virus technique” J. Immunol. 127:1275-1280 (1981); Traggiaiet al., “An efficient method to make human monoclonal antibodies frommemory B cells: potent neutralization of SARS coronavirus” Nat. Med.10:871-875 (2004); and Winter et al., “Man-made antibodies” Nature349:293-299 (1991). Furthermore, protocols for isolating antibody heavyand light chain variable region (VH and VL) complementary DNA pairs byRT-PCR directly from single B lineage cells have also been designed.Lagerkvist et al., “Single, antigen-specific B cells used to generateFab fragments using CD40-mediated amplification or direct PCR cloning”Biotechniques 18:862-869 (1995); Babcook et al., “A novel strategy forgenerating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined specificities” Proc. Natl. Acad. Sci.USA 93:7843-7848 (1996); and Meijer et al., “Isolation of human antibodyrepertoires with preservation of the natural heavy and light chainpairing” J. Mol. Biol. 358:764-772 (2006). Although feasible, thesesystems are limited by the throughput for the selection ofantigen-specific B cells. A cell-based high-throughput method has beenreported for detecting and recovering individual antigen-specificantibody secreting cells using a microwell array chip that can analyzeup to 234,000 individual cells (i.e. a polyclonal mixture of primaryhuman lymphocytes) at once. Jin et al., “A Rapid And EfficientSingle-Cell Manipulation Method For Screening Antigen-SpecificAntibody-Secreting Cells From Human Peripheral Blood” Nature Method 1-6(2009). This method has been term “immunospot array assay on a chip”wherein a conventional microarray chip (230K microwell array) compriseswells coated with generalized anti-immunoglobulin antibodies. If anantibody secreting cell is placed in these wells a distinct circularspot formed by the binding of target antigen to the specific antibodies.The antibody-secreting cells were then recovered using amicromanipulator (TransferMan NK2, Eppendorf) fitted with capillaries(Primetech) under the fluorescence microscope and then were expelledinto microtubes for reverse transcription. The cDNA was then used toproduce recombinant antibody.

The high functionality of an integrated microfluidic chip can lead tothe realization of small instruments and disposable devices due to highsensitivity and low cost. In particular, by combining mechanical,physical, and sometimes electrical principles, microfluidic chips haveevolved to carry out the manipulation of small numbers ofmicroparticles, or sometimes a single particle, such as a cell, on asmall chip. The dynamic monitoring of a single cell in an independentlycontrolled environment is important in eliminating the influences byother cells such as a mixture of hormones, ions, and neurotransmittersreleased from the neighboring cells. To date, several technologies havebeen reported for the manipulation of single micro particle on amicrofluidic chip including: i) physical capturing (Huang et al.,“Transport, location, and quantal release monitoring of single cells ona microfluidic device” Anal. Chem. 76:483-488 (2004); Thielecke et al.,“Fast and precise positioning of single cells on planar electrodesubstrates” IEEE Eng. Med. Biol. Mag. 1848-52 (1999); Wheeler et al.,“Microfluidic device for single-cell analysis” Anal. Chem. 75:3581-3586(2003); and Yun et al., “Micro/nanofluidic device for single-cell-basedassay” Biomed. Microdevices 7:35-40 (2005)); ii) cell sorting (Fu etal., “A microfabricated fluorescence-activated cell sorter” Nat.Biotechnol. 17:1109-1111 (1999); and Shirasaki et al., “On-chip cellsorting system using laser-induced heating of a thermoreversiblegelation polymer to control flow” Anal. Chem. 78:695-701 (2006)); iii)optical tweezers (Ashkin et al., “Optical trapping and manipulation ofsingle cells using infrared laser beams” Nature 330:769-771 (1987); Araiet al., “High-speed separation system of randomly suspended singleliving cells by laser trap and dielectrophoresis” Electrophoresis22:283-288 (2001); and Grier D., “A revolution in optical manipulation”Nature 424:810-816 (2003)); iv) dielectrophoresis (Müller et al., “A 3-Dmicroelectrode system for handling and caging single cells andparticles” Biosens. Bioelectron. 14:247-256 (1999); Hughes, “Strategiesfor dielectrophoretic separation in laboratory-on-a-chip systems”Electrophoresis 232569-2582 (2002); Manaresi et al., “A CMOS chip forindividual cell manipulation and detection” IEEE J. Solid State Circuits38:2297-2305 (2003); and Taff et al., “A scalable addressablepositive-dielectrophoretic cell sorting array” Anal. Chem. 77:7976-7983(2005)); v) electric field-driven capturing (Toriello et al.,“Microfluidic device for electric field driven single-cell capture andactivation” Anal. Chem. 77:6935-6941 (2005)); and vi) optoelectrictweezers (Chiou et al., “Massively parallel manipulation of single cellsand microparticles using optical images” Nature 436:370-372 (2005)). Butin most cases, these techniques fail to provide all of the functionsrequired for a general bioassay, which include but are not limited to,the isolation of each chamber, chemical stimulation, and/or themonitoring of reactions in a fast and highly parallel manner. Amultifunctional microwell plate in the form of a microfluidic chip withmultiple microwells in a two-dimensional array for high-throughput cellanalysis and drug screening has been reported using three PDMS layersand a silicon substrate to create micro-wells that physically isolatecaptured cells so that specific reagent(s) can be introduced into eachmicro-well without continuously maintaining a cell suspension mediaflow. Yun et al. “Multifunctional Microwell Plate For On-Chip Cell andMicrobead-Based Bioassays” Sensors and Actuators B 143: 387-394 (2009).Specifically, the bottom PDMS layer is etched with a pattern ofmicro-wells and microchannels for inlet, outlet and drug injectionchannels. The middle PDMS layer forms a cover that seals themicro-wells. The top PDMS layer forms a connection port to a vacuumchamber that actuates the middle PDMS cover flap. The cover flapprovided a tight seal such that cross-contamination between microwellsis prevented. Each microwell has its own dedicated chemical injectionand drain channel on the bottom side, which can induce a chemicalreaction in the captured bio-materials or provide chemical excitation ofthe cells located in the designated target microwells by the selectiveinjection of specific reagents. In this manner, each set of cells withineach microwell can be individually exposed to different environmentalconditions (i.e., for example, stimulatory and/or inhibitory hormones)without affecting the cells in a neighboring microwell. The device isnot configured to recover the cells after entry and is therefore limitedto bio-assays, including the capturing of bio-materials into multiplemicrowells, well isolation, and/or the introduction of specificchemicals.

Most conventional biochemical assays are performed using a considerablenumber of cells to determine their quantitative biomolecular profiles,such bulk assays only provide their averaged values in the analyzedensemble and thus often overlook important information regarding theirfluctuations among individual cells. Ferrells et al., Science280:895-898 (1998); and Levsky et al., Trends Cell Biol. 13:4-6 (2003).Some believe that to perform quantitative analysis of intracellularbiological contents at the single-cell level must integrate cell lysisfollowed by a quantitative analysis of biochemical contents in thelysate. For example, a multiplexed single-cell analyses method reportedthus far take advantage of highly sophisticated and automatedinstruments for integration of these multiple steps on a singleplatform, e.g., single-cell capture followed by chemical lysis in aclosed volume of 50 pL recently reported in a microfabricated device.Irimia et al., Anal. Chem. 76:6137-6143 (2004). However, the complicatedflow path and process of the microfabricated device in that report couldbe a serious drawback for extending the application to single-cellbiochemistry. A single-cell lysis method has been reported for analyzingintracellular content and enzymatic activity at the cellular level usinga dense array of microwells (10-30 picoliter) fabricated in PDMS.Experimental assays on single cells isolated within these microwellsdemonstrate the ability to detect proteins by antibody conjugatedmicrobeads as well as protease activity by fluorescent substrates.Sasuga et al. “Single-Cell Chemical Lysis Method For Analyses OfIntracellular Molecules Using An Array Of Picoliter-Scale Microwells”Analytical Chemistry 80(23): 9141-9149 (2008). As this method inherentlyresults in the lysis of the evaluated cells, they cannot be recoveredfor future use after identification of their biochemical content.

Optical cell manipulation methods centered around optical tweezers havebeen adapted to cell sorting and are intuitive wherein a user directlyfocuses a laser onto a target cell and uses the beam to tweeze or pushthe cell to a desired location. Ashkin et al., Optics Letters 11:288-290(1986): and Buican et al., Applied Optics 26:5311-5316 (1987). Opticaltweezer arrays and optical lattices can optically manipulate and sortmultiple cells and particles simultaneously. Grier D G., Nature424:810-816 (2003); and MacDonald et al., Nature 426:421-424 (2003).However, the high-numerical aperture (NA) requirements of opticaltweezers greatly restrict imaging field size and constrain devicearchitecture due to the short working distances of the objective lensestypically used. While a small field size is sufficient to work withlarge numbers of small particles or small cells such as bacteria, onlysmall numbers of mammalian cells can fit in such a small field (˜2500μm²) for optical tweezer array-based manipulation. Further, powerrequirements are high, as each trap site might require upwards of ˜100mW of optical power Optoelectronic tweezers (OETs) employ lower-NAoptics, and thus enable larger area (˜1 mm2) manipulation fields viaoptically mediated dielectrophoretic (DEP) trap arrays, extending“virtual” optical manipulation to field areas better suited formammalian cell manipulation. Chiou et al., Nature 436:370-372 (2005).Unfortunately, owing to buffer incompatibilities, OET forces exerted oncells suspended in standard cell culture medium are weak, and a ˜1 mm²manipulation area, while an improvement over traditional opticaltweezers, is still insufficient to simultaneously manage largepopulations of cells. Use of a large-area display to directly actuateOETs without lenses circumvents the issue of lens field size, butdecreases manipulation resolution and suffers from the same bufferincompatibilities of traditional OETs. Choi et al., Microfluidics andNanofluidics 3:217-225 (2007). Array-based systems employing non-opticalconfinement methods can form arrays of cells extending beyond a singleimaged field. DEP trap arrays have successfully demonstrated trap andrelease sorting capability and can, in principle, be scaled to largearray sizes. Taff et al., Analytical Chemistry 77:7976-7983 (2005).Unfortunately, such site-addressable electrical approaches requirecomplex on-chip interconnects and significant off-chip support circuitrywhen scaled to large array sizes. Hydrodynamic trap arrays, utilizingeither microwells or obstacles for cell confinement, offer simple,passive, mostly single-cell loading over large areas with minimalcomplexity, allowing microscopy-based imaging of large arrays over timeto investigate single-cell behavior. Rettig et al., Analytical Chemistry77:5628-5634 (2005); and Di Carlo et al., Lab on a Chip 6:1445-1449(2006). Viable retrieval of small numbers of single cells from microwellarrays using micropipettes/micromanipulators based on temporalfluorescence behavior has also been demonstrated, but the retrievalmethod is time-consuming and cumbersome. Love et al., NatureBiotechnology 24:703-707 (2006); and Yamamura et al., AnalyticalChemistry 77:8050-8056 (2005). A microscope-compatible microfluidiccell-sorting device has been reported that contains a microwell arraythat is passively loaded with mammalian cells via sedimentation prior tovisual inspection by microscopy. Kovac et al. “Intuitive, Image-BasedCell Sorting Using Opto-Fluidic Cell Sorting” Analytical Chemistry79(24):9321-9330 (2007). A PDMS microwell array is molded from a siliconwafer master that produces 105 μm flow channels and 25-30 μm diameterposts to pattern a microwell array. This array supports over 10,000individually addressable trap sites. A glass slide was then bonded tothe microwell array to complete the formation of a sealed chamber. Aninfrared laser is then focused upon a single cell (i.e. within a singlemicrowell) resulting in actuation and levitation from the microwellsinto a flowing media for collection. This method is limited toimage-based cell sorting and the devices are not configured to detectany secreted biochemical compounds into the media solution. Othermicroarray methods that isolate biological cells including but notlimited to: i) a multi-analyte biosensor chip comprising electrodes forelectrical measurement of analytes (Saleh et al., “Direct Detection ofAntibody-Antigen Binding Using An On-Chip Artificial Pore” PNAS 100(3):820-824 (2003)); ii) measuring cellular responses (i.e. drug testing,toxicology and basic cell biology) using phase-contrast and fluorescencemicrographs (Rettig et al. “Large-Scale Single-Cell Trapping And ImagingUsing Microwell Arrays” Analytical Chemistry 77(17): 5628-5634 (2005));iii) microwell cell culture substrates capable of cultivating hundredsto thousands of individual cell cultures (Charnley et al., “IntegrationColumn: Microwell Arrays For Mammalian Cell Culture” Integrative Biology1: 625-634 (2009)); iv) single cell analysis platform for microscopicanalysis, on-chip fluorescent assays and enzyme kinetics (Di Carlo etal., “Single-Cell Enzyme Concentration, Kinetics, and InhibitionAnalysis Using High-Density Hydrodynamic Cell Isolation Arrays”Analytical Chemistry 78(14): 4925-4930 (2006)); v) detecting singleantigen specific B cells based on fluctuations in antigen-inducedintracellular Ca²⁺ immobilization and/or fluorescence-labeled antigenbinding (Kinoshita et al., “Identification Of Antigen-Specific B CellsBy Concurrent Monitoring Of Intracellular Ca2+ Mobilization And AntigenBinding With Microwell Array Chip System Equipped With A CCD Imager”Cytometry Part A 75A(8): 682-687 (2009)); vi) a hydrogel microwell fordynamically studying the fate of single cells by time-lapse microscopy.“Regulation Of Stem Cell Fate In Bioengineered Arrays Of HydrogelMicrowells” California Institute for Regenerative Medicine,cirm.ca.gov/node/46.

II. Screening Problems Solved by the Direct Cloning and SelectionTechnology

Conventionally used screening display methods (i.e., for example, phagedisplay) have numerous technical challenges, including, but not limitedto, size of protein displayed has to be small, multiplicity of infection(MOI) needs to be high to avoid loss of diversity, is dependent on theactivity of the phage, technically challenging and needs highly trainedpeople, multiple panning rounds needed (taking up to 1 week or more),high non-specific binding due to phage, antibodies may not function wellin soluble form (truncated clones are often expressed), and/or avidityeffects can hinder selection of high affinity clones.

A generalized overview of conventional high throughput processes foridentifying interaction pairs in parallel is described below. See, FIG.14 Starting with a source of DNA (usually millions of different copies),proteins are translated (either in vitro or in vivo) from the DNA andlinked to a screening substrate. Through multiple rounds of screening,specific binders are separated from background non-specific binders.Target clones are subsequently recovered and re-amplified via theirgenotype-phenotype linkage. The most commonly used methods are the‘display’ methods, especially phage display. Traditionally, spatialaddressing methods can be simple and efficient, but are limited by thenumbers of clones that be analyzed. For the first time, the presentinvention provides a spatial addressing technique that match phage andcell display technologies in cell number but with greater efficiency ina shorter time.

In particular, conventional bacteriophage display screening techniqueshave specific disadvantages including but not limited to requiring thedisplay of the protein, high non-specific binding levels (i.e.,providing a low signal-to-noise ratio), or a prolonged period of time inwhich to run the assay (i.e., for example, fourteen days). On the otherhand, direct clone analysis and selection has specific advantages overthese conventional screening methods, including, but not limited to:recovery of the cells is not limited by a display of a particularprotein, low non-specific binding level (i.e., providing a highsignal-to-noise ratio), short period of time in which to run the assay(i.e., for example, two days), highly parallel and scalable therebyallowing the testing of millions or billions of recombinant antibodiesin a single cycle; a pore-based array which has distinct advantages overwell-based arrays, a very high density micro-pore array for screeningbiological interactions, selection of biological material and cellsusing a commercially available micro-pore array (manufactured by thebonding of millions or billions of silica capillaries and the fusingthem together through a thermal process); screening millions (orbillions) of biological interactions in parallel; independent recoveryof millions of target cells; provides qualitative concentration versusaffinity information for millions of clones in parallel. (e.g. feedbackon production efficiency is provided for each expressed gene);eliminates the non-specific binding due to phage display; truemonovalent binding, simultaneous testing of at least two (or more)different antigens per pore (e.g. simultaneous positive and negativescreening); significant reductions in assay time (i.e., for example, for14 days, to 1-2 days), significant reduction in costs of new antibodydiscovery; and/or does not require complex biological procedures, thusis be more reproducible and robust.

The present invention is capable of processing a number of cells that isorders of magnitude greater that any known screening technique. Thepresent invention is similar to ribosome, phage display, andmicroengraving only to the point of collecting DNA, and transforming thecells with the collected DNA. Both ribosomal and phage selectionprocesses require multiple rounds (i.e., bio-panning) to enrich for thecells of interest, while microengraving is limited by both the depth ofthe wells and the number (˜10⁵). See, FIG. 19A, Step 1. The presentinvention is capable of selecting for interactions between binding pairshaving an improved specificity over known screening techniques thatcannot handle the rapid and error-free extraction of thousands (or more)of cells. For example, the repeated nature of ribosomal and phagedisplay panning inherently results in the arbitrary loss of specifictypes of binding pairs, while microengraving is limited tomicromanipulator capture techniques thereby inducing error and recoverylosses to recover specific cells. For example, such microengravingtechniques cannot handle the fast and error free extraction of 1000s ormore cells. See, FIG. 19B, Step 2. In summary, the direct clone analysisand selection technique described herein provides advantages in thelargest total screened cell number in the shortest possible time. See,FIG. 19C, Summary.

III. Methods of Making Direct Cloning and Selection Arrays

Microarrays have been created by sectioning bundles of small plasticrods, fibers, tubes or tubules wherein biological components (i.e.nucleic acid fragments, nucleotides, antigens, antibodies, proteins,peptides, carbohydrates, ligands, receptors, drug targets or biologicalcells) are bound to (i.e., immobilized) the sides of the rods or fibersduring their manufacture. Anderson et al. “Microarrays And TheirManufacture By Slicing,” U.S. Pat. No. 7,179,638 (herein incorporated byreference). These microarrays that are coated with biological componentsare used to perform a variety of different quantitative biochemicalanalyses such as enzymatic activities, immunochemical activities,nucleic acid hybridization and small molecule binding. These immobilizedbinding components may be coated on the inside or outside of microtubes,contained in a gel that is placed within the microtubes, orattached/embedded in small particles or beads that fill the tubes. Whenthe individual fibers are solid rods or filaments, the bindingcomponents may be incorporated on the rod of filament surface, orimpregnated within the filament during casting of a filament block.Consequently, each coated microarray section that is cut from the sameblock constitutes a coated microarray for use in the same bindingassays. For example, a block that is a meter long can be cut into10-micron thick sections thereby yielding 100,000 identical coatedmicroarrays.

Unlike the micro-pore array of the present invention, the coatedmicroarray may also have specific fibers incorporated with a solidifyingmedium (i.e., for example, a hydrogel or bead) attached to the bindingcomponents prior to filling the hollow fibers thereby creating amini-matrix to support biochemical reactions. Further, the coatedmicroarrays filled with a supporting medium by using hydrostatic forceor centrifugal force, rather than a superior, but optional, method ofusing a degassed solid substrate utilized by embodiments of the presentinvention as described herein.

Generally, the biological cells and/or biochemical reactants areimmobilized within individual fibers prior to slicing off the coatedmicroarrays, such that the cells/reactants are retained inside thehollow fibers after the microarray is formed. Generally, the celldensity introduced into each fiber exceeds 1 million cells per squarecentimeter, but when using smaller fibers microarrays may comprise up toat least 10 billions cells per square centimeter of the array. Thesepre-filled coated microarrays are intended for long-term storage for usein analysis and detection assays. The coated microarrays are notcompatible with the detection of in vivo secretion of biological agentsfrom freshly cultivated biological cells.

Unlike the micro-pore array of the present invention, the coatedmicroarrays are then attached directly between at least two adhesivesurfaces, flexible films or solid surfaces to produce microarray chips,such that the coated microarray is sandwiched in between the two solidsubstrates. These coated microarrays might be used for cloning ofbiological cells, viruses or other particles by adding dilutesuspensions to the microarray but they are incompatible with the directcloning and selection technology described herein.

The micro-pore arrays contemplated herein can be manufactured bybundling millions or billions of silica capillaries and fusing themtogether through a thermal process. Such a fusing process may comprisethe steps including but not limited to; i) heating a fiber single drawglass that is drawn under tension into a single clad fiber; ii) creatinga fiber multi draw single fiber from the single draw glass by bundling,heating, and drawing; iii) creating a fiber multi-multi draw multi fiberfrom the multi draw single fiber by additional bundling, heating, anddrawing; iv) creating a block assembly of drawn glass from themulti-multi draw multi fiber by stacking in a pressing block; v)creating a block pressing block from the block assembly by treating withheat and pressure; and vi) creating a block forming block by cutting theblock pressing block at a precise length (i.e., for example, 1 μm). See,FIG. 15. In one embodiment, the method further comprises slicing thesilica capillaries, thereby forming a very high-density glass micro-porearray plate. It will be appreciated that the array of micro-pores foruse in the present invention can be formed by any suitable method, aslong as the internal diameter of the micro-pores ranges betweenapproximately 1.0 micrometers and 500 micrometers. In one embodiment,the capillaries are cut to approximately 1 millimeter in height, therebyforming a plurality of micro-pores having an internal diameter betweenapproximately 1.0 micrometers and 500 micrometers. In one embodiment,the micro-pores range between approximately 10 micrometers and 1millimeter long. In one embodiment, the micro-pores range betweenapproximately 10 micrometers and 1 centimeter long. In one embodiment,the micro-pores range between approximately 10 micrometers and 10millimeter long. In one embodiment, the micro-pores range betweenapproximately 10 micrometers and 100 millimeter long. In one embodiment,the micro-pores range between approximately 0.5 millimeter and 1 meterlong.

Such processes form a very high-density micro-pore array that is used inthe present invention. In some arrays, each micro-pore comprises a 5 μmdiameter and an approximate 66% open space. In some arrays, the array is10×10 cm and comprises over 300 million micro-pores. See, FIG. 1. Insome arrays, the proportion of the array that is open (i.e., comprisesthe lumen of each micropore) ranges between about 50% and about 90%, forexample about 60 to 75%, such as a micro-pore array provided byHamamatsu and having an open area of about 67%.

The internal diameter of micro-pores ranges between approximately 1.0micrometers and 500 micrometers. In some arrays, each of saidmicro-pores can have an internal diameter in the range betweenapproximately 1.0 micrometers and 300 micrometers; optionally betweenapproximately 1.0 micrometers and 100 micrometers; further optionallybetween approximately 1.0 micrometers and 75 micrometers; still furtheroptionally between approximately 1.0 micrometers and 50 micrometers,still further optionally, between approximately 5.0 micrometers and 50micrometers.

In some arrays, the open area of the array comprises up to 90% of theopen area (OA), so that, when the pore size varies between 10 μm and 500μm, the number of micro-pores per cm² of the array varies between 458and 1,146,500, as is represented in the table below. In some arrays, theopen area of the array comprises about 67% of the open area, so that,when the pore size varies between 10 μm and 500 μm, the number ofmicro-pores per cm² of the array varies between 341 and 853,503, as isrepresented in the table below. It will be appreciated that, with a poresize of 1 μm and up to 90% open area, each cm² of the array willaccommodate up to approximately 11,466,000 micro-pores.

Pore diameter (um) No of pore (90% OA) No of pores (67% OA) 500 458 341300 1275 948 100 1150 8,535 75 20,380 15,172 50 45,860 34,140 101,146,500 853,503 1 11,465,967 85,350,318

In one embodiment, the method further comprises coating a solidsubstrate with a binding partner. In one embodiment, the binding partnercomprises an antigen. In one embodiment, the solid substrate comprisesPDMS. In one embodiment, the method further comprises degassing thesolid substrate to activate a degassed driven flow (i.e., for example,for approximately fifteen minutes). In one embodiment, the methodfurther comprises placing the glass micro-pore array plate on thedegassed solid substrate to create a degassed testbed array. See, FIG.2. Although it is not necessary to understand the mechanism of aninvention, it is believed that degassing the solid substrate results inself-powered pumping to load the glass micro-pore array plate with amedia solution. Degassing of the solid substrate is, however, not neededto load the glass micro-pore array plate with a media solution. See,FIG. 27.

In one embodiment, the present invention contemplates a method forloading a degassed testbed array comprising contacting a solutioncomprising a plurality of cells with the degassed testbed array to forma loaded testbed array. Generally, degassing of a solid substrate, suchas PDMS, is performed by placing the substrate in a vacuum chamber forone to three hours. A degassed substrate (i.e. PDMS) permits the removalof gas, such as air bubbles for example, from the substrate as well asthe overlying capillaries that may hinder or prevent direct interactionsbetween antigen immobilized on the substrate surface and antibodypresent in the media. The capillaries attached to a sufficientlydegassed substrate may be loaded with sample for a period of timefollowing the degassing step. In some instances the sample may be loadedinto the capillaries an hour (or more) after the degassing step has beenperformed. The substrate may be degassed multiple times if necessary.For example, the degassing step may be repeated if the sample is notloaded quickly enough following the initial degassing step.

In another embodiment, the present invention contemplates a method forloading a testbed array comprising contacting a solution comprising aplurality of cells with the testbed (non-degassed) array to form aloaded testbed array.

In one embodiment, loading a mixture of antibody secreting E. colievenly into all the micro-pores comprises placing a 500 μL droplet onthe upper side of the array and spreading it over all the micro-pores.The heterologous population of cells can be loaded onto the micro-porearray. If a solid substrate is present, the heterologous population ofcells can be loaded onto the micro-pore array prior to reversibleattachment of the micro-pore array with the solid substrate.Alternatively, if a solid substrate is present, the heterologouspopulation of cells can be loaded onto the micro-pore array afterreversible attachment of the micro-pore array with the solid substrate.In one embodiment, an initial concentration of approximately 10⁹ cellsin the 500 μL droplet results in approximately 3 cells (orsub-population) per micro-pore. In one embodiment, each micro-pore hasan approximate volume of between 20-80 pL (depending on the thickness ofthe glass capillary plate of between 250 μm to 1 mm). Once themicro-pores are loaded and incubated overnight, each micro-pore shouldthen contain approximately 2,000-3,000 cells per micro-pore. In oneembodiment, the cells may be cultivated for up to forty-eight hourswithout loss of viability in order to maximize the proliferation yield.Although it is not necessary to understand the mechanism of aninvention, it is believed that “spreading” the droplet over all themicro-pores provides for optimal distribution of cells in the variousmicro-pores. Theoretically, adding a drop to the micro-pore array shouldfill all pores evenly. However, an empirical evaluation demonstratedthat surface tension actually prevents the drop from entering thecentral micro-pores. See, FIG. 20. If the drop is spread evenly over themicro-pore array surface the surface tension is removed. See, FIG. 21.Consequently, if the drop is placed straight down on the micro-porearray, only the pores at the edge of the drop fill due to reducedsurface tension (also evaporation recedes the drop so that the liquid isno longer held in suspension). This causes a halo ring effect followingdetection of the appropriate analyte. See, FIGS. 22A, 22B and 22C. Inone embodiment, the solution comprises approximately three (3)microliters. In one embodiment, the plurality of cells may be selectedfrom the group comprising animal cells, plant cells, and/or microbialcells. In one embodiment, the plurality of cells comprise E. coli cells.In one embodiment, the E. coli cells secrete at least one recombinantcompound of interest. In one embodiment, the recombinant compound ofinterest has an affinity for the binding partner. Although it is notnecessary to understand the mechanism of an invention, it is believedthat, if there are approximately 10⁹ cells in an approximate 500 μLsolution then, on average, there should be approximately three (3) cellsper micro-pore for an array having approximately 3-4×10⁶ micro-pores. Itshould be noted that the exact number will depend on the number of poresin the array. For example, if an array has approximately 3-4×10⁶micro-pores, it therefore, would have approximately 500-100 cells/pore.In one embodiment, each micro-pore comprises a volume of ranging betweenapproximately 20-80 picoliters.

IV. Methods of Using Direct Cloning and Selection Arrays

The data described herein demonstrate that superior antigen-specificpositive signal is obtained when soluble antibody is produced byon-plate culturing rather than in test tubes. Results furtherdemonstrate that the high degree of non-specific binding that occursduring phage display is totally eliminated when the antibody isselectively expressed in soluble form from specific cells that arecompartmentalized within a micro-pore. In addition, the use of wholephage particles leads to poor resolution due to their large sizerelative to the displayed antibody. Thus, in addition to being fasterand easier to use, the ability to detect secreted antibody allows thismicro-pore array to provide higher resolution than current methods thatrely upon the target molecule being expressed on the surface of adisplay vector (i.e. phage display, ribosome display, mammalian celldisplay, bacterial cell display or yeast display). Additional benefitsof this array as compared to phage display methods include the abilityto simultaneously test two (or more) target molecules per pore (i.e.positive and negative screening) and not being limited by the size ofthe protein being examined since phage-displayed proteins have to besmall.

A. Biological Recognition Assays

Although it is not necessary to understand the mechanism of aninvention, it is believed that the testbed arrays described abovecomprise micro-pores having sufficient volume to incubate the cells forbetween 0-48 hours, such that compounds of interest are secreted fromthe cells and bind to the binding partner. Consequently, a plurality ofbiological recognition assays may be performed either within, orbetween, each of the micro-pores. For example, one such recognitionassay may comprise antigen-antibody binding.

In one embodiment, the present invention contemplates a method forantigen-antibody binding comprising incubating a plurality of cells at37° C. for 1-24 hrs such that each cell produces antibodies and secretesthe antibodies into the micro-pore. In one embodiment, the antibody is arecombinant antibody. In one embodiment, the antibody is a monoclonalantibody. In one embodiment, at least one of the cells produces morethan one antibody. Although it is not necessary to understand themechanism of an invention, it is believed that because of the micro-porearchitecture, this incubation is relatively free of evaporation lossesdue to the very narrow inlets (thus small exposed surface area) and theextreme length of the pores. In one embodiment, the antibody secretionis stimulated by an induction agent. In one embodiment, the inductionagent comprises IPTG. See, FIGS. 4A and 4B.

In one embodiment, the present invention contemplates an in-solutionbinding assay, wherein each binding partner is in solution orsuspension. Such binding partners include, but are not limited to:Bimolecular fluorescence complementation (BiFC); Protein-Fragment;Complementation Assays (PCA) such as with Split ubiquitin,β-Galactosidase, β-Lactamase, Luciferase, Dihydrofolate Reductase andGreen Fluorescent Protein); Yeast two hybrid (Y2H); Bacterial twohybrid; Tandem affinity purification (TAP); Fluorescence resonanceenergy transfer (FRET); Bioluminescence resonance energy transfer(BRET); Homogeneous fluorescence polarization assay; AmplifiedLuminescent Proximity Homogeneous Assay; Homogeneous Caspases Assay;Back-Scattering Interferometry (BSI) and Particle-based systemsfluorescent or plasmonic systems.

B. Cell Isolation and Selection

In one embodiment, the present invention contemplates a method forisolating and selecting a cell within a micro-pore. In one embodiment,the method comprises separating the binding partner-coated solidsubstrate from the micro-pore array. In one embodiment, the bindingpartner-coated solid substrate comprises an antigen-primary antibodycomplex. In one embodiment, the method further comprises incubating theseparated binding-partner-coated solid substrate with a secondarylabeled anti-tag antibody to detect the antigen-primary antibodycomplex. In one embodiment, the detected antigen-primary antibodycomplex forms a detectable spot. See, FIG. 5. In one embodiment, thedetectable spot is fluorescent. In one embodiment, the detectable spotis radioactive. In one embodiment, the detectable spot is spin-labeled.In one embodiment, the solid substrate comprising the labeledantigen-primary antibody complex is scanned to locate high intensitybinding spots by spatial addressing. See, FIG. 6. Although it is notnecessary to understand the mechanism of an invention, it is believedthat the scanning locates the specific micro-pore address comprisingcells secreting the compound of interest (i.e., for example, thespecific antibody).

In one embodiment, the method further comprises isolating the spatiallyaddressed cells located in the micro-pores corresponding to the highintensity binding spots. In one embodiment, the isolating may beselected from the group comprising pressure ejection, degas driven flow,and/or electrolytic expulsion. In one embodiment, the isolated cell isextracted, cultured and identified for recovery of the cell DNA.

In one embodiment, the method further comprises isolating cells locatedin the micro-pores by pressure ejection. For example, a separatedmicro-pore array is covered with a plastic film. In one embodiment, themethod further provides a laser capable of making a hole through theplastic film, thereby exposing the spatially addressed micro-pore. See,FIGS. 17 and 17 a. In FIGS. 17 and 17A, the bright circular feature inthe centre is the 40 μm hole and the other circular features are animprinted image of the array left on the scotch tape. Subsequently,exposure to a pressure source (i.e., for example, air pressure) expelsthe contents (i.e., for example, cells) from the spatially addressedmicro-pore. See, FIG. 18. In one embodiment, the hole is betweenapproximately 500 μm-1 μm. In one embodiment, the hole is betweenapproximately 100 μm-5 μm. In one embodiment, the hole is betweenapproximately 50 μm-10 μm. In one embodiment, the hole is between 25μm-15 μm. In one embodiment, the laser is a non-melting laser.

FIG. 18A demonstrates single pore removal from a 40 μm pore diametermicropore filter array. A 40 μm diameter micropore filter array wasfilled with food colourant (dark pores) and the contents of one singlepore removed by focussed air pressure through a 40 μm hole in scotchtape that sealed the whole array.

C. Therapeutic Drug Discovery

In one embodiment, the present invention contemplates a methodcomprising identifying new therapeutic drugs. For example, a solidsubstrate may be coated with a drug binding partner known to be involvedin a disease condition (i.e., for example, a biological receptor and/orenzyme) or a non-immobilized drug binding partner known to be involvedin a disease condition is provided. A plurality of cells secretingvarious compounds suspected of having affinity for the binding partneris then screened using the very high-density micro-pore array. Themicro-pores containing the binding partner-compound complexes having thehighest affinity are selected for future development.

D. Diagnostic Antibody Discovery

In one embodiment, the present invention contemplates a methodcomprising identifying diagnostic antibodies. For example, a solidsubstrate may be coated with a binding partner known to be involved in adisease condition (i.e., for example, an antigen and/or epitope) or anon-immobilized drug binding partner known to be involved in a diseasecondition is provided. A plurality of cells secreting various antibodiessuspected of having affinity for the binding partner is then screenedusing the very high-density micro-pore array. The micro-pores containingthe binding partner-antibody complexes having the highest affinity areselected for future development.

E. Protein-Protein Interaction Studies

In one embodiment, the present invention contemplates a methodcomprising identifying protein-protein interactions. For example, asolid substrate may be coated with a binding partner known to beinvolved in a disease condition (i.e., for example, a protein and/orpeptide) or a non-immobilized drug binding partner known to be involvedin a disease condition is provided. A plurality of cells secretingvarious proteins and/or peptides suspected of having affinity for thebinding partner is then screened using the very high-density micro-porearray. The micro-pores containing the binding partner-protein or peptidecomplexes having the highest affinity are selected for futuredevelopment.

F. Protein-Nucleic Acid Interaction Studies

In one embodiment, the present invention contemplates a methodcomprising identifying protein-nucleic acid interactions. For example, asolid substrate may be coated with a binding partner known to beinvolved in a disease condition (i.e., for example, a deoxyribonucleicacid and/or a ribonucleic acid and/or a SOMAmer and/or a Apatamer) or anon-immobilized drug binding partner known to be involved in a diseasecondition is provided. A plurality of cells secreting various proteinsand/or peptides suspected of having affinity for the binding partner isthen screened using the very high-density micro-pore array. Themicro-pores containing the binding partner-nucleic acid complexes havingthe highest affinity are selected for future development.

G. Protein-Carbohydrate Interaction Studies

In one embodiment, the present invention contemplates a methodcomprising identifying protein-carbohydrate interactions. For example, asolid substrate may be coated with a binding partner known to beinvolved in a disease condition (i.e., for example, an oligosaccharide,and liposaccharide, or a proteosaccharide) or a non-immobilized drugbinding partner known to be involved in a disease condition is provided.A plurality of cells secreting various lectins, proteins and/or peptidessuspected of having affinity for the binding partner is then screenedusing the very high-density micro-pore array. The micro-pores containingthe binding partner-carbohydrate complexes having the highest affinityare selected for future development.

V. Kits

In another embodiment, the present invention contemplates a kitcomprising: a first container comprising an array of micro-pores,wherein the internal diameter of micro-pores range between approximately1.0 micrometers and 500 micrometers; and a second container comprisingat least one binding partner. In one embodiment the second containercomprises a solid substrate comprising said at least one bindingpartner, the solid substrate being capable of reversible attachment tothe array of micro-pores.

In another embodiment, the present invention contemplates kits for thepractice of the methods of this invention. The kits preferably includeone or more containers containing a micro-pore array comprising aplurality of fused capillary fibers that are not coated with a pluralityof binding partners. The kit can optionally include a solid substratecomprising a plurality of binding partners. The kit can optionallyinclude a plurality of labeled reagents capable of detecting a varietyof binding partner-biological compound complexes. The kit can optionallyinclude a solution comprising a biological cell comprising a recombinantprotein. The kits may also optionally include appropriate systems (e.g.opaque containers) or stabilizers (e.g. antioxidants) to preventdegradation of the reagents by light or other adverse conditions.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the micro-porearray in the detection of various biological compounds that are secretedfrom a biological cell. While the instructional materials typicallycomprise written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated by this invention. Such media include, but arenot limited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

VI. Antibodies

The present invention provides recombinant antibodies (i.e., forexample, polyclonal or monoclonal). In one embodiment, the presentinvention provides monoclonal antibodies that specifically bind to avariety of antigens and/or epitopes. These antibodies find use in thedetection methods described above.

An antibody against a protein of the present invention may be anymonoclonal or polyclonal antibody, as long as it can recognize theprotein. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animalwhose antibody titer has been confirmed (e.g., a mouse) is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter and antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and the like.The proportion of the number of antibody producer cells (spleen cells)and the number of myeloma cells to be used is preferably about 1:1 toabout 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added inconcentration of about 10% to about 80%. Cell fusion can be carried outefficiently by incubating a mixture of both cells at about 20° C. toabout 40° C., preferably about 30° C. to about 37° C. for about 1 minuteto 10 minutes.

Various methods may be used for screening for a hybridoma producing theantibody (e.g., against a tumor antigen or autoantibody of the presentinvention). For example, where a supernatant of the hybridoma is addedto a solid phase (e.g., microplate) to which antibody is adsorbeddirectly or together with a carrier and then an anti-immunoglobulinantibody (if mouse cells are used in cell fusion, anti-mouseimmunoglobulin antibody is used) or Protein A labeled with a radioactivesubstance or an enzyme is added to detect the monoclonal antibodyagainst the protein bound to the solid phase. Alternately, a supernatantof the hybridoma is added to a solid phase to which ananti-immunoglobulin antibody or Protein A is adsorbed and then theprotein labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification. Normally, a medium for animal cells towhich HAT (hypoxanthine, aminopterin, thymidine) are added is employed.Any selection and growth medium can be employed as long as the hybridomacan grow. For example, RPMI 1640 medium containing 1% to 20%, preferably10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetalbovine serum, a serum free medium for cultivation of a hybridoma(SFM-101, Nissui Seiyaku) and the like can be used. Normally, thecultivation is carried out at 20° C. to 40° C., preferably 37° C. forabout 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO₂gas. The antibody titer of the supernatant of a hybridoma culture can bemeasured according to the same manner as described above with respect tothe antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody can be carried outaccording to the same manner as those of conventional polyclonalantibodies such as separation and purification of immunoglobulins, forexample, salting-out, alcoholic precipitation, isoelectric pointprecipitation, electrophoresis, adsorption and desorption with ionexchangers (e.g., DEAE), ultracentrifugation, gel filtration, or aspecific purification method wherein only an antibody is collected withan active adsorbent such as an antigen-binding solid phase, Protein A orProtein G and dissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. Material containing theantibody is recovered from the immunized animal and the antibody isseparated and purified.

As to the complex of the immunogen and the carrier protein to be usedfor immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

In addition, various condensing agents can be used for coupling of ahapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like,of an animal immunized by the above method. The antibody titer in theantiserum can be measured according to the same manner as that describedabove with respect to the supernatant of the hybridoma culture.Separation and purification of the antibody can be carried out accordingto the same separation and purification method of immunoglobulin as thatdescribed with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, a protein expressed resultingfrom a virus infection (further including a gene having a nucleotidesequence partly altered) can be used as the immunogen. Further,fragments of the protein may be used. Fragments may be obtained by anymethods including, but not limited to expressing a fragment of the gene,enzymatic processing of the protein, chemical synthesis, and the like.

VII. Detection Methodologies

A. Detection of Nucleic Acids

mRNA expression may be measured by any suitable method, including butnot limited to, those disclosed below.

In some embodiments, RNA is detection by Northern blot analysis.Northern blot analysis involves the separation of RNA and hybridizationof a complementary labeled probe.

In other embodiments, RNA expression is detected by enzymatic cleavageof specific structures (INVADER assay, Third Wave Technologies; Seee.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and5,994,069; each of which is herein incorporated by reference). TheINVADER assay detects specific nucleic acid (e.g., RNA) sequences byusing structure-specific enzymes to cleave a complex formed by thehybridization of overlapping oligonucleotide probes.

In still further embodiments, RNA (or corresponding cDNA) is detected byhybridization to an oligonucleotide probe. A variety of hybridizationassays using a variety of technologies for hybridization and detectionare available. For example, in some embodiments, TaqMan assay (PEBiosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and5,538,848, each of which is herein incorporated by reference) isutilized. The assay is performed during a PCR reaction. The TaqMan assayexploits the 5′-3′ exonuclease activity of the AMPLITAQ GOLD DNApolymerase. A probe consisting of an oligonucleotide with a 5′-reporterdye (e.g., a fluorescent dye) and a 3′-quencher dye is included in thePCR reaction. During PCR, if the probe is bound to its target, the 5′-3′nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probebetween the reporter and the quencher dye. The separation of thereporter dye from the quencher dye results in an increase offluorescence. The signal accumulates with each cycle of PCR and can bemonitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used todetect the expression of RNA. In RT-PCR, RNA is enzymatically convertedto complementary DNA or “cDNA” using a reverse transcriptase enzyme. ThecDNA is then used as a template for a PCR reaction. PCR products can bedetected by any suitable method, including but not limited to, gelelectrophoresis and staining with a DNA specific stain or hybridizationto a labeled probe. In some embodiments, the quantitative reversetranscriptase PCR with standardized mixtures of competitive templatesmethod described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978(each of which is herein incorporated by reference) is utilized.

B. Sequencing of Nucleic Acids

The method most commonly used as the basis for nucleic acid sequencing,or for identifying a target base, is the enzymatic chain-terminationmethod of Sanger. Traditionally, such methods relied on gelelectrophoresis to resolve, according to their size, wherein nucleicacid fragments are produced from a larger nucleic acid segment. However,in recent years various sequencing technologies have evolved which relyon a range of different detection strategies, such as mass spectrometryand array technologies.

One class of sequencing methods assuming importance in the art are thosewhich rely upon the detection of PPi release as the detection strategy.It has been found that such methods lend themselves admirably tolarge-scale genomic projects or clinical sequencing or screening, whererelatively cost-effective units with high throughput are needed.

Methods of sequencing based on the concept of detecting inorganicpyrophosphate (PPi) which is released during a polymerase reaction havebeen described in the literature for example (WO 93/23564, WO 89/09283,WO98/13523 and WO 98/28440). As each nucleotide is added to a growingnucleic acid strand during a polymerase reaction, a pyrophosphatemolecule is released. It has been found that pyrophosphate releasedunder these conditions can readily be detected, for exampleenzymatically e.g. by the generation of light in theluciferase-luciferin reaction. Such methods enable a base to beidentified in a target position and DNA to be sequenced simply andrapidly whilst avoiding the need for electrophoresis and the use oflabels.

At its most basic, a PPi-based sequencing reaction involves simplycarrying out a primer-directed polymerase extension reaction, anddetecting whether or not that nucleotide has been incorporated bydetecting whether or not PPi has been released. Conveniently, thisdetection of PPi-release may be achieved enzymatically, and mostconveniently by means of a luciferase-based light detection reactiontermed ELIDA (see further below).

It has been found that dATP added as a nucleotide for incorporation,interferes with the luciferase reaction used for PPi detection.Accordingly, a major improvement to the basic PPi-based sequencingmethod has been to use, in place of dATP, a dATP analogue (specificallydATP.alpha.s) which is incapable of acting as a substrate forluciferase, but which is nonetheless capable of being incorporated intoa nucleotide chain by a polymerase enzyme (WO98/13523).

Further improvements to the basic PPi-based sequencing technique includethe use of a nucleotide degrading enzyme such as apyrase during thepolymerase step, so that unincorporated nucleotides are degraded, asdescribed in WO 98/28440, and the use of a single-stranded nucleic acidbinding protein in the reaction mixture after annealing of the primersto the template, which has been found to have a beneficial effect inreducing the number of false signals, as described in WO00/43540.

C. Detection of Protein

In other embodiments, gene expression may be detected by measuring theexpression of a protein or polypeptide. Protein expression may bedetected by any suitable method. In some embodiments, proteins aredetected by immunohistochemistry. In other embodiments, proteins aredetected by their binding to an antibody raised against the protein. Thegeneration of antibodies is described below.

Antibody binding may be detected by many different techniques including,but not limited to, (e.g., radioimmunoassay, ELISA (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotope labels,for example), Western blots, precipitation reactions, agglutinationassays (e.g., gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to cancer markers isutilized.

In other embodiments, the immunoassay is as described in U.S. Pat. Nos.5,599,677 and 5,672,480; each of which is herein incorporated byreference.

D. Remote Detection Systems

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given marker or markers) into data ofpredictive value for a clinician. The clinician can access thepredictive data using any suitable means. Thus, in some preferredembodiments, the present invention provides the further benefit that theclinician, who is not likely to be trained in genetics or molecularbiology, need not understand the raw data. The data is presenteddirectly to the clinician in its most useful form. The clinician is thenable to immediately utilize the information in order to optimize thecare of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, wherein the information is provided to medicalpersonal and/or subjects. For example, in some embodiments of thepresent invention, a sample (e.g., a biopsy or a serum or urine sample)is obtained from a subject and submitted to a profiling service (e.g.,clinical lab at a medical facility, genomic profiling business, etc.),located in any part of the world (e.g., in a country different than thecountry where the subject resides or where the information is ultimatelyused) to generate raw data. Where the sample comprises a tissue or otherbiological sample, the subject may visit a medical center to have thesample obtained and sent to the profiling center, or subjects maycollect the sample themselves (e.g., a urine sample) and directly sendit to a profiling center. Where the sample comprises previouslydetermined biological information, the information may be directly sentto the profiling service by the subject (e.g., an information cardcontaining the information may be scanned by a computer and the datatransmitted to a computer of the profiling center using an electroniccommunication systems). Once received by the profiling service, thesample is processed and a profile is produced (i.e., expression data),specific for the diagnostic or prognostic information desired for thesubject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment for the subject, along with recommendationsfor particular treatment options. The data may be displayed to theclinician by any suitable method. For example, in some embodiments, theprofiling service generates a report that can be printed for theclinician (e.g., at the point of care) or displayed to the clinician ona computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease.

E. Detection Kits

In other embodiments, the present invention provides kits for thedetection and characterization of proteins and/or nucleic acids. In someembodiments, the kits contain antibodies specific for a proteinexpressed from a gene of interest, in addition to detection reagents andbuffers. In other embodiments, the kits contain reagents specific forthe detection of mRNA or cDNA (e.g., oligonucleotide probes or primers).In preferred embodiments, the kits contain all of the componentsnecessary to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results.

EXPERIMENTAL Example I Glass Pore Array Feasibility Study

A fused silica micro-pore array was obtained having regularly spacedmicro-pores with a 10 μm diameter, and a 66% total open area, thus thereis a very high density distribution of micro-pores (i.e., for example,between approximately 3 and 5 million). See, FIGS. 7A and 7B.

Example 1a

Bacteriophage assay: A PDMS layer (solid substrate) was first coatedwith bacteriophage particles, followed by placement of theabove-mentioned micro-pore array (array having regularly spacedmicro-pores with a 10 μm diameter and a 66% total open area) on the PDMSlayer. Different regions of the assembled micro-pore testbed array wereloaded with solutions comprising different concentrations of biotinlabeled anti-phage antibodies. Alternatively, the micro-pore array canbe loaded with the biotin labeled anti-phage antibody solution and thenplaced on the PDMS layer. It is preferred that the micro-pore testbedarray is assembled by pacing the micro-pore array on the PDMS layer,after which the biotin labeled anti-phage antibody solution is appliedonto the micro-pore testbed array.

The micro-pore array was separated from the PDMS layer and the PDMSlayer was stained with FITC labeled Extravidin, thereby selectivelystaining the biotin-labeled anti-phage antibodies.FITC signals were seen to vary in proportion with the phage-boundantibody concentration. See, FIG. 8. Micropore filter array andmicrotitre plate immunoassay comparison with human IgG (antigen) andgoat anti-human IgG Cy5-labelled polyclonal antibody. See FIG. 8 a.

Example 1b

Human IgG competitive immunoassay: A competitive assay was performedunder identical conditions on both a Nunc microtitre plate and on theassembled micro-pore testbed array comprising PDMS sealed microporefilter array. Human IgG was coated on the surface and decreasingconcentrations of human IgG in solution mixed 1:1 (v:v) with a setconcentration of goat anti-human IgG Cy5-labelled antibody.

The aim of this example is to show that antibody-antigen bindinginteractions can be performed in the micropore array and that theimmunoassay sensitivity the array assay is comparable with that of astandard microliter plate assay.

Materials

Hamamatsu capillary plate filter (Hamamatsu J5022-16)PDMS (Sylgard 184 Silicone Elastomer Kit, Dow Corning order No:(400)000104984061)

Bovine Serum Albumin, BSA (Sigma A9418)

Bacteriophage particles (New England Biolabs M13KO7 helper phage, N0315)Biotin labeled anti-fd bacteriophage antibody (Sigma B2661)

Extravidin-FITC (Sigma E2761) Human IgG (Biomeda Corp Cat No:MS143)

Goat anti-human IgG polyclonal antibody, Cy5 labeled (Biomeda Corp CatNo:SJ15147-C)

Methods Example 1a: Bacteriophage Assay

-   -   The PDMS solid substrate was prepared by mixing 10 parts of        elastomer with 1 part curing agent followed by curing at 60° C.        for 2 hours. PDMS slabs were prepared to between 3 and 5 mm in        thickness and cut to size as needed so that the micro-pore array        could be placed on top.    -   The PDMS slab was coated with M13KO7 helper phage at 1×10⁹        phage/mL in PBS for 1 hour at 37° C. after which it was        submerged in 10 mL PBS containing 3% (w/v) BSA for 1 hour at 37°        C.    -   The coated and blocked PDMS slab was washed with 5 mL of PBS and        5 mL of ultrapure water and subsequently air dried and degassed        by placing the slab in a vacuum chamber consisting of a plastic        desiccator linked up to vacuum tap at approximately 1 to 1×10⁻³        Torr for 45 minutes.    -   The micro-pore array (glass capillary array) was placed on top        of the degassed PDMS slab and pressed tightly against the slab        to form a reversible liquid-tight seal, preventing any possible        leakage.    -   Immediately, three microliters of neat biotinylated anti-fd        bacteriophage antibody, 1/100 antibody diluted in PBS and        1/10,000 dilution of antibody diluted in PBS were added to 3        different sections of the glass micropore array using a        micropipette and spread evenly over the micropores. Spreading        the liquid after placing on to the array prevents the ‘halo        effect (see FIGS. 20, 21 and 22A-C) that can occur due to        surface tension forces that are present when the sample is left        as a droplet on the array.    -   The PDMS slab/micropore array was incubated at 37° C. for 1        hour.    -   To visualize binding on the PDMS slab, the glass array was        manually peeled off and the PDMS slab washed with 10 mL of PBST        (PBS with 0.05% Tween 20). Eight hundred microliters of a 1/50        dilution of Extravidin FITC was placed on top of the PDMS slab        covering the analysis area. After incubation for 1 hour at 37°        C., the slab was washed with 20 mL of PBST, air dried and        visualized using a fluorescent microscope with FITC filter.

Example 1b: Human IgG Competitive Immunoassay

-   -   Eight hundred microliters of a five micrograms per mL of human        IgG diluted in PBS was used to coat the surface of a PDMS slab.        100 μL/well of the five micrograms per mL of human IgG diluted        in PBS was used to coat the wells of a black Nunc Maxisorb 96        well microtiter plate, Each were incubated for 1 hour at 37° C.    -   Both the PDMS slab and the microtiter plate were blocked with 3%        (w/v) BSA in PBS (200 μL/well for the microtiter plate while the        slab was submerged in 5 mL) for 1 hour at 37° C.    -   Goat anti-human IgG Cy5-labelled polyclonal antibody diluted        1/1000 in PBS was mixed 1:1 (v/v) with decreasing concentrations        of human IgG (final concentrations ranged from 60 to 0.2        micrograms per mL) and subsequently added to wells of the        microtiter plate (100 μL/well) and to specific regions of the        micro-pore array (3 μL of each dilution added to the array).    -   The array was then immediately placed on top of the degassed        antigen coated and blocked PDMS slab and reversibly sealed by        applying manual pressure.    -   Both the array and the microtiter plate were incubated at 37° C.        for 1 hour before being washed with PBST (20 mL PBST was passed        over the slab while each well of the microtiter plate was washed        four times).    -   The fluorescent intensity from bound Cy5 labeled antibody in the        microtiter plate was measured using a Tecan Safire2 plate reader        (Ex. 650, Em. 670) while the fluorescent intensity on the PDMS        slab was measured using a PerkinElmer microarray scanner with        Cy5 filter        Conclusion: The experiments described here show that, not only        can an immunoassay be performed in the glass micropore array        reversibly attached to PDMS but, despite the extremely low        volume per pore (78 pL), excellent immunoassay sensitivity        correlation with the standard 96 well plate format is achieved.

Example II Micro-Pore Cell Viability

This example demonstrates the viability and expression capability ofgreen fluorescent protein-expressing E. coli incubated in a micro-porearray.

Example IIa

Verification that E. coli cells can enter the micro-pores of the array:E. coli cells were loaded onto a micro-pore testbed array comprising themicro-pore array as described in Example I placed on an uncoated andunblocked PDMS layer, the micro-pore testbed array then being incubatedfor 1.5 hrs. The PDMS layer was then separated whereby the data showthat E. coli cells remained on the PDMS post culture and could bevisualized on the PDMS surface using a fluorescent microscope. See,FIGS. 9A and 9B.

Example IIa

Verification that E. coli cells can express recombinant protein in themicro-pores: E coli cell viability in the micro-pore testbed array wasdemonstrated with a GFP induction assay, where non-GFP E. coli cellswere induced to produce GFP when loaded into the micro-pores by mixingthe cells with an induction medium. GFP fluorescence within each wellwas recorded in real time. Heat inactivated cells (10 minutes at 70° C.)were used as a negative control. See, FIG. 10.

Aim: To show that E. coli cells can enter the 10 μm diameter pores andexpress recombinant protein in the micro-pores

Materials

E. coli Rosetta cells (Novagen) transformed with a pET28b plasmid(Novagen) containing the gene encoding for enhanced green fluorescentprotein (sequence identical to eGFP from pEGFP accession number U76561)Fluorescent microscope with automated stage and camera (OLYMPUS IX81)Hamamatsu capillary plate filter (Hamamatsu J5022-16)PDMS (Sylgard 184 Silicone Elastomer Kit, Dow Coming order No:(400)000104984061)

Methods Example IIa: Verification that E. coli Cells Can Enter theMicro-Pores

-   -   E. coli Rosetta cells transformed with a pET28b plasmid        containing the gene encoding for enhanced green fluorescent        protein were grown in 2×TY media until an O.D.600 of 0.5 was        reached at which time IPTG (Fisher BPE-1755-10) was added to a        final concentration of 1 mM. The culture was further incubated        for 4 hours at 30° C. with agitation at 200 rpm.    -   The culture was then diluted 1/100 in 2×TY media and 5 μL of        neat and 1/100 culture added to the micropore array of Example I        reversibly bound to an uncoated and unblocked degassed PDMS        slab. The array was incubated for 1.5 hours at 37° C. before the        array was gently removed and the PDMS visualized using a        fluorescent microscope.

Example IIa: Verification that E. coli Cells Can Express RecombinantProtein in the Micro-Pores

-   -   E. coli Rosetta cells transformed with a pET28b plasmid        containing the gene encoding for enhanced green fluorescent        protein were grown in 2×TY media until an O.D.600 of 0.5 was        reached at which time the cultures were pelleted by        centrifugation (3300 g for 10 minutes) and the pellet        resuspended in fresh 2×TY media to the initial culture volume.    -   Five milliliters of culture was transferred to two sterile        universal tubes and one (positive culture) was kept at 37° C.        for 10 minutes and the other (negative culture) was placed in a        70° C. water bath for 10 minutes.    -   After 10 minutes, IPTG (Fisher BPE-1755-10) was added to a final        concentration of 1 mM to each tube, mixed by inverting and 5 μL        of each culture added to separate regions of a 10 μm diameter        micro-pore array.    -   The micro-pore array was reversibly attached to an unmodified        PDMS slab and cultured in an incubation chamber attached to an        automated fluorescent microscope for 4 hours at 37° C.    -   The microscope with automated stage was programmed to take        images of each culture region every 5 minutes. The intensity of        the fluorescence emitted from the expressed GFP from both        cultures is shown in FIG. 10 and exemplary frames are visualized        in FIG. 10B.

Conclusion: E. coli cells were easily loaded into the 10 μm diametermicropore array and expressed an abundance of recombinant GFP.Expression of GFP from one single micro-pore from the heat-killedculture indicates that one cell (high probability that only one cellsurvived as no other pore contained cells that survived) survived andthis could be easily and clearly detected.

Example III Antibody Secretion from E coli Cells

This example demonstrates that cells can secrete (i.e., leak) antibodiesinto a culture media such that binding can occur at an antigen coatedsolid surface.

Example IIIa

Two antibody clones (anti-Halofuginone (HFG) scFv and anti-prostatespecific antigen (PSA) scFv) were grown in micro-pores coated withHFG-Transferin, PSA and BSA. Both clones were expected to only bindtheir cognate antigens and not to the other two surfaces. In addition,the anti-HFG clones were expressed as both soluble antibody and on thesurface of phage to evaluate the effects of phage particles.

All 3 clones (soluble and phage anti-HFG and soluble anti-PSA) were alsogrown in 20 mL tubes overnight and added to a microtiter plate thefollowing day and incubated for 1 hour. Similar results would have beenexpected following incubation on a micro-pore test bed array. Theresults showed that a larger antigen specific signal was observed whensoluble antibody was produced from culturing on the microtiter plate ascompared to that of the same clones grown in conventional test tubes.See, FIG. 11.

Further, the scFv phage display was observed to have a high degree ofnon-specific binding. In fact, during a normal screening run, an scFvantibody clone would probably not be selected due to the highnon-specific binding which is totally eliminated when the antibody isexpressed in soluble form.

Example IIIb

Analysis of E. coli cells harbouring scFv antibody genes to C-reactiveprotein (CRP) and cardiac Troponin I (cTnI): E. coli cultures containingscFv genes were added to different sections of a micro-pore array (10 μmpores) and cultured for 24 hours. Cells propagated and expressed scFvantibody fragments into the media that could interact with their cognateantigen that was pre-coated on the PDMS surface (column 1, labelledCoated-Cells). See FIG. 25.

The same cultures were also added to parts of the array which was notcoated with antigen (Non-coated Cells, column 3) and media added to bothcoated and non-antigen coated PDMS section through the array (columns 2and 4).

FIG. 25 demonstrates that specific recombinant antibody response can bedetected when cultured in the array.

Aim: To show that E. coli cells expressing recombinant single chainfragment variable (scFv) antibodies secrete/leak antibody into the mediawithout the need for secretion signals and this antibody can be detectedwhen bound to coated solid support.

Materials Example IIIa

On plate culture of antibodies and subsequent detection of expressedantibody

Antigens

PSA (Lee Biosolutions, Missouri, USA, Cat. No. 497-11)Halofuginone-BTG (was purchased from Chris Elliot, The Queen'sUniversity of Belfast, Northern Ireland)CRP (Life Diagnostics Inc, PA, USA. Cat. No. 8000)cTnI (Life Diagnostics Inc, PA, USA. Cat. No. 1210)

Antibodies—Examples IIIa and IIIb

Recombinant avian scFv antibodies were developed and selected asdescribed herein. RNA was extracted from the spleens and bone marrow oftwo chickens immunised with target analyte in TRI-Reagent andfirst-strand cDNA synthesis performed using the Superscript III kit(Invitrogen). Antibody variable heavy and light chain genes wereamplified using the primer sets described by Andris-Widhopf andco-workers (“Methods for the generation of chicken monoclonal antibodyfragments by phage display”, Andris-Widhopf J, Rader C, Steinberger P,Fuller R, Barbas CF 3rd., J Immunol Methods. 2000 Aug.28;242(1-2):159-81) and cloned into the pComb3× vector (kind gift fromthe Barbas lab, San Diego (The Skaggs Institute for Chemical Biology andthe Departments of Molecular Biology and Chemistry, The Scripps ResearchInstitute, 10550 North Torrey Pines Rd., La Jolla, Calif. 92037) in aVL-VH scFv format, with a 18 amino acid flexible linker joining bothvariable domains and a HA tag for detection and antibody capture. ClonedscFv genes were electroporated into E. coil XL-1 blue (Strategene, LaJolla, Calif. 92037) cells generating an antibody library ofapproximately 3×10⁷ clones. The scFv fragments were packaged on thesurface of M13K07 phage and subjected to four rounds of panning againstmicrotitre plate wells (Maxisorp, Nunc) coated with 10, 5, 1 and 1 □g/mltarget antigen, respectively. After panning, eluted phage werere-infected into E. coil TOP10F′ (Invitrogen) cells and single coloniesselected for monoclonal ELISA in sterile 96 well culture plates. ScFvproduction was induced by the addition of 1 mM IPTG overnight at 30° C.prior to screening for binding to antigen in solution (competitiveELISA).

Example IIIa: Method

For a direct method comparison between antibodies grown in pre-coatedELBA plate wells and antibodies grown in culture and later added toantigen coated microtiter plate wells, two scFv fragments and one scFvclone rescued on the surface of M13KO7 helper phage were inoculated intoa 50 mL sterile tubes containing 10 mL of SB broth supplemented with 50μg/mL carbenicillin and 10 μg/mL tetracycline. These were grown whileshaking at 37° C. until the O.D.600 reached 0.8, at which time IPTG wasadded to a final concentration of 1 mM. Immediately after induction, 200μL of culture was added to an antigen-coated ELISA plate (pre-coatedwith 1 μg/mL PSA, 1 μg/mL HFG-BTG or PBS and blocked with 3% (w/v) BSA)wells and both the ELISA plate and 50 mL culture flask incubatedovernight together at 30° C. with constant agitation. The following day,200 μL of ‘off-plate’ samples (samples cultured in 50 mL tubes) werealso added directly to the ELISA plate. All samples were incubated at37° C. for a further 1 hour and detected with anti-HA HRP antibody andTMB substrate.

Method: Example IIIb

Analysis of E. coli cells harbouring scFv antibody genes to C-reactiveprotein (CRP) and cardiac Troponin I (cTnI) cultured in a 10 μm diametermicropore array. Antibody cultures were prepared by transferring 10 μLfrom −80° C. glycerol stocks of E. coli cells containing pComb3×plasmids encoding anti-CRP or anti-cTnI scFv antibody genes to 990 μLauto induction media. The cultures were mixed by inversion and 5 μL ofeach culture added to two regions on separate 10 μm diameter microporearrays. Media was also added as a negative control. The 10 μm diametermicropore array was subsequently reversibly attached to a PDMS slabcoated with cognate antigen on one region and blocking buffer only onthe other region so that each antibody culture was in contact withantigen and blocking only as an additional negative control. Afterincubation at 37° C. overnight, bound antibody was detected with anti-HACy5-labelled antibody using a PerkinElmer microarray scanner.

Example IV Cell Removal from the Micro-Array Testbed

This example demonstrates that cells cultured in the 10 μm diametermicro-array testbed for 24 hrs at 37° C. can be extracted by applyingair pressure to the testbed and recovered on agar plates. Growth of thecells on agar plates following further incubation at 37° C. demonstratescell viability.

The experiment was performed to confirm cell growth and viability in thearray after 24 hours incubation at 37° C. E. coli cells harbouring thepET28b plasmid encoding the GFP gene fused to a cTnI peptide were grownto an OD₆₀₀˜0.4 at which time they were induced for 1 hour to allow GFPexpression. The cells were diluted in media to 1/10, 1/100, 1/1000 and1/10000 and 2 μL of each dilution (including the neat culture and mediaonly as a negative control) added to a section of the array. The arraywas sandwiched between two pieces of PDMS (not degassed) and left at 37°C. for 24 hours. Meanwhile, 100 μL/well of the each dilution was alsoadded to a black Nunc plate and the fluorescence measured. The plate wasleft overnight at 37° C. shaking and the fluorescence measured thefollowing day to determine cell growth. See FIG. 12. Cells from the openperforated glass array were recovered onto agar plate by blowing theliquid out using a pipette tip linked to compressed air tube. The plateswere incubated overnight to confirm that the cells in the array werestill viable after 24 hours. See FIG. 13.

Conclusion: The contents of a micropore can be expelled by applicationof a gas source. In addition, this demonstrates that, when the cells areexpelled onto agar, the cells are still viable.

Example V Precision Liquid Removal from the Micro-Array Testbed

This example demonstrates that small volumes of liquid can be removedfrom precise positions in the micro-array testbed (10 μm diameter) byplacing a cover over the array which contains a small (100 μm diameter)hole.

A black box (approximately 1 cm by 2 cm) was printed on to a Xeroxtransparent sheet (Type A, P/N 003R96019) using a standard laserprinter. The box was cut out and placed into the focus of a high powerlaser, which melted a circular 100 μm diameter hole in the sheet. SeeFIG. 17. The pores in the micro-array testbed were filled with 3 μL ofred food colorant to aid visualization. The section of printed blacktransparent sheet with the 100 μm hole was placed over the food colorantfilled testbed array and clamped in position. A tube (approximately 2-3mm in diameter) connected to a compressed air source was placed over thehole and air pressure applied. The air was forced to flow through thehole and r the food colorant was removed (expelled) from the testbedarray. Food colorant was removed in a circular pattern to a diameter of300 μm, 3 times the size of the hole used. See FIG. 18.

Example VI Antibody Secretion from B Cells

B cells (B lymphocyte cell line, ATCC No: TIB-196 (Designation U266,supplier: LGC Standards, Middlesex, UK) (about 1×10⁴ cells/mL) wereadded to a 40 μm pore diameter array, cultured for 4 days and expressedantibody analysed by sandwich ELISA. The bright dots represent theantibody patterned PDMS from single pores that had B cells expressingantibody. The spots are 40 μm in diameter. See FIG. 23.

FIG. 23 demonstrates that specific B cell antibody response can bedetected when cultured in the array and individual positive pores can bevisualized. The background represents those pores that had no B cellsexpressing antibody or did not contain any B cells at all. Without beingbound by theory, it is thought that, when a B cell suspension of about1×10⁴ cells/mL is distributed across micropores having an internaldiameter of 40 μm and a volume of 1.2 nL, there may about 1 cell inapproximately every 84 micropores.

Analysis of B cell culture grown and screened in the micropore filterarray. B cells were added to a 40 μm pore diameter array, cultured for 4days and expressed antibody analysed by sandwich ELISA. See FIG. 24.

The fluorescence of individual B cells pores was compared to that ofpores with no cells or pores with B cells not expressing antibody. FIG.24 illustrates that specific B cell antibody response can be detectedwhen cultured in the array.

Sandwich ELISA for the detection of human IgE secreted from U266 B cellline:

Coated 2 μg/mL Mab 107 (Mabtech code 3810-3-250) (capture antibody)Detected with 0.33μg/mL Mab 182-biotin antibody diluted with PBS with0.5% (w/v) BSA for 1 hour followed by the addition ofstreptavidin-dylight 650 (Fisher Cat. No. 84547) diluted in PBS with0.5% (w/v) BSA to a final concentration of 2 μg/mL.

Example VII Non-Degassed Driven Loading and Patterning of the MicroporeArray

Goat anti-human IgG Cy5 antibody was loaded in to the capillary array(10 μm diameter pores) and sealed with unmodified PDMS that was notdegassed. A clear array pattern was observed indicating that degasdriven loading of the solid substrate (for example, PDMS) is notcritical for the device, kit and method of the present invention. SeeFIG. 26 a.

This experiment shows that substantially even patterning using a dyelabeled protein can be achieved without degassing. FIG. 26b shows adirect comparison in assay between degassed and non-degassed loading. Itwill be appreciated that the choice of degassing v non-degassing thesolid substrate is independent of choice of binding partners, forexample, the antibody-antigen pair—in either case, there must be liquidcontact with the solid substrate (for example, PDMS) surface.

A direct comparison of a direct binding immunoassay using degassed andnon-degassed antigen coated PDMS was also performed. Two PDMS slabs werecoated with 5 μg/mL of human IgG and blocked with 3% (w/v) BSA. Goatanti-human IgG Cy5-labelled antibody diluted to 50, 5 and 0.5 μg/mL inPBS was added to different regions on both arrays and incubated for 1hour at 37° C. Bound antibody was detection using a PerkinElmermicroarray scanner. See, FIG. 26 b.

Example VIII Determination of Enhanced Surface Coating with APTES CoatedPDMS

Direct binding assay. Goat anti-human IgG Cy5 antibody was added tohuman IgG coated PDMS that had been modified with APTES treatment (leftpicture) or unmodified (right picture). Both signal and uniformity isenhanced with APTES treatment. See, FIG. 27. APTES (Sigma, A3648-100mLs)

-   -   3% (v/v) APTES was prepared in 98% ethanol    -   PDMS slab was exposed to O2 plasma (a procedure to clean or        modify the surface of materials, such as PDMS, by using an        energetic plasma created from gaseous species, such as argon and        oxygen, or gaseous mixtures such as air and hydrogen/nitrogen        for 6 minutes before being submerged in 3% APTES solution for 1        hour at 25° C.    -   Slab washed with ethanol    -   Slab dried by baking at 60° C. for 2 hours before being coated        with antigen of interest.

Example X Single Pore Removal Setup and Procedure

Using an in house built high precision dual XY-stage and laser setup,cells residing in target pores may be harvested into wells of a 384 wellplate by laser ablation of array sealant and subsequent liquid removalby application of air pressure. See FIGS. 28a, 28b and 28 c.

Aim: To show that E. coli cells can be retrieved from single micro-poresand that the cells are viable once recovered in growth media.

E. coli cells containing a pET26b plasmid and enhanced GFP gene werecultured overnight in SB (Super Broth, contents/liter: 35 g tryptone, 20g yeast extract, 5 g NaCl) growth medium containing 25 μg/mL, kanamycinand 1% (w/v) glucose. The next day, 60 μL of SB containing 25 μg/mLkanamycin was added to wells of a 384 well plate and 1 μL of a 1/100dilution of overnight cells added to one well of the 384 well as apositive control. The plate was added to the lower XY stage as shown inFIG. 28a . Next, 25 μL of the overnight E. coli culture was added to a40 μm diameter micropore array. The array was sealed with scotch tape(˜55 μm in thickness), loaded onto the array holder and the holdersubsequently attached to the upper XY stage. The laser/air nozzleassembly was lowered to 2 mm above the array while the bottom surface ofthe array sat less than 0.5 mm above the well plate. Two single holesover above a cultured filled pore were ablated in situ along with cellextraction using nitrogen (with a power setting of 8% from a 25 W laserfor approximately 0.5 seconds). The pore contents were extracted into amedia filled well of the 384 plate underneath the area and the XY stagemoved to capture cells in different wells of the 384 well plate asneeded. One well with media only was used as a negative control wherebyno cells were added to that well. The 384 well plate was incubated at37° C. for 18 hours and then the O.D.600 measured using a Tecan Safire2microtiter plate reader. The absorbance values of each well are shown inFIG. 18 b.

Results

FIG. 18b : Single pore recovery of viable E. coli cells from a 40 μmdiameter micropore array. Panel (I) shows two ˜40 μm holes ablated inscotch tape (3M Company, ˜55 μm in thickness) which is sealed to a 40 μmdiameter micro-pore array (supplied by Incom USA). The contents of eachmicro-pore (cells) are instantaneously removed once the hole is ablateddue to the nitrogen flow from the laser nozzle. Panel (II) shows thesame micro-pore array with the scotch tape removed, revealing two emptypores at the precise location of the ablated holes. Panel (III) shows agraph representing cell viability after being recovered from a singlepore in to a single well of a 384 well microtiter plate containing 60μL, of growth media. Pore 1 is the left-hand empty pore of panel (II)and pore 2 is the right-hand empty pore of panel (II). The positivecontrol sample represents a well containing 60 μL of media spiked with 1μL of overnight E. coli culture and the negative control represents 60μL of media only.

Conclusion: Using the cell recovery setup of the present invention, E.coli cells from a single pore of a micropore array can be recovered intoa well of a 384 well plate containing growth media and cultured toexpand the cell population.

Example XI In Solution Assays—Demonstration of GFP Reassembly in theArray

Aim: The aim of this experiment was to demonstrate an in-solutionbinding assay in the array, using the GFP reassembly vectors describedby Magliery et al. (J Am Chem Soc. 2005 Jan. 12;127(1):146-57.“Detecting protein-protein interactions with a green fluorescent proteinfragment reassembly trap: scope and mechanism”. Magliery T J, Wilson CG, Pan W, Mishler D, Ghosh I, Hamilton A D, Regan L). This system usesthe reassembly of the dissected fragments of GFP to identify specificprotein-protein interactions. Vectors were obtained from the authors andthe positive control vectors were used for the proof of conceptexperiments, described herein.

Details of System

Vector 1:pET11a-Z-NGFP (NZ) contains the N-terminal region of GFP fusedto a leucine zipper peptide (anti-parallel to that on Vector 2). It isampicillin resistant and expression is induced by IPTG addition (Lac Ioperon).Vector 2:pMRBAD-Z-CGFP (CZ) encodes the C-terminal of GFP fused to adifferent leucine zipper peptide (anti-parallel to that on Vector 1). Itis kanamycin resistant and expression is induced by Arabinose addition(AraC operon).

Method

Plasmids were transformed into BL21 (DE3) E.coli separately. Asequential transformation was then carried out, meaning that the cellstransformed with one plasmid were made competent and then transformedwith the second plasmid. This meant that both plasmids were containedwithin the same cell, denoted in this report as “in vivo”.

Positive transformants were identified by growth on screening mediawhich contains 1 μM IPTG, 0.2% Arabinose, 35 μg/mL Kanamycin, 100 μg/mLCarbenicillin. This was carried out on solid media and also in solution(in plate and on array). Cultures were grown at 37° C. overnight,followed by 2-3 days at 15-25° C.

In addition to ‘in vivo’ screening, cells containing single plasmidswere mixed and co-expressed, denoted in this report as ‘in vitro’. Thiswas done in the absence of antibiotics, as growth would have beeninhibited. A 10 μL aliquot of each of the following, in appropriateexpression media, was added to the array and incubated for 5 days at 25°C.

-   -   (a) CZ only    -   (b) NZ only    -   (c) CZ+NZ ‘in vivo’ (i.e. in the same cell)    -   (d) CZ+NZ ‘in vitro’ (i.e. mixed culture)

Results ‘In Vivo’ Screening

Following 2.5 days of expression on screening media, the level offluorescence was observed in a transilluminator box, wavelength ˜365 nm.FIG. 29: Demonstration of reassembly of GFP ‘in vivo’ in liquid media.Cells have been pelleted by centrifugation at 4000 rpm for 10 mins andthe supernatant removed.

‘In Vitro’ Screening—On Array

The level of fluorescence was compared in three ways:

1. Visualisation using a transilluminator (not illustrated herein).

2. Visualisation using a microscope (not illustrated herein)

3. Measurement of the fluorescence intensity from images using histogramfunction in Image J as demonstrated in FIG. 30. FIG. 30: Graphicalrepresentation of the fluorescent intensity values obtained frommicroarray pore array with E. coli cultures harbouring split GFPfragments using a fluorescent microscope.

Example XII In Solution Assays—FRET Demonstration

Experiment: Demonstration of in solution DNA binding assay in themicropore array using FRET.Aim: The aim of this experiment was to demonstrate an in-solutionbinding assay in the array, using the FRET detection based on thereassembly two complimentary DNA probes labelled with Cy5 and Cy5.5.Once DNA hybridisation occurs, the Cy5 and Cy5.5 dyes are brought inclose proximity with each other so that fluorescence energy transfer canoccur between the Cy5 donor and Cy5.5 acceptor. Due to the transfer ofenergy, the intensity of emitted light from Cy5 is reduced in thepresence of bind Cy5.5 held in close proximity by the bound DNA.

Details of System

DNA probe 1: 5′-Cy5-TTACggTTggTggCgTCTCTg DNA probe 2:5′-AATgCCAAC CAC CgCAgA gAC-Cy5.5Both DNA probes were purchased from TIB MOLBIOL, Berlin, Germany.

Method

In order to measure the energy transfer between the donor and acceptorin solution, 1×10⁻⁸M solutions of the donor labeled oligonucleotide wasmixed 1:1 (v/v) with hybridisation buffer alone or with hybridisationbuffer containing 1×10⁻⁸M acceptor labelled complementaryoligonucleotide. Acceptor labelled oligonucleotide was also mixed 1:1(v/v) with buffer results in three final solutions containing donoralone, donor and acceptor mixed and acceptor alone. 100 μL of eachsolution was immediately added to wells of a black Nunc 96 well plateand 2 μL of each solution added to different regions of a 40 μm diametermicropore arrays. Both array and micotitre were incubated at 37° C. for30 mins before analysis. The micotitre plate was analysed using aTecanStaphire II (Ex 610 nm and em 650-750 scan) fluorescent platereader and the array analysed using a fluorescent microscope with Cy5filter.

Results

DNA-FRET was successfully demonstrated using fluorescence intensitymeasurements from spectral analysis in a microtitre plate (FIG. 31) andin a 40 μm diameter micropore array (FIG. 32). Binding of the twocomplementary DNA probes is detected by a clear decrease in Cy5 emissiondue fluorescent energy transfer with the Cy5.5 acceptor.

1. A device comprising an array of micro-pores, the micro-pore arraybeing reversibly attached to a solid substrate, wherein at least onebinding partner is attached to said solid substrate, and wherein theinternal diameter of the micro-pores ranges between approximately 1.0micrometers and 500 micrometers.
 2. The device of claim 1, wherein themicro-pores are not coated with at least one binding partner. 3-4.(canceled)
 5. The device of claim 1, wherein said device furthercomprises a polymeric film, wherein said array is covered by saidpolymeric film, wherein said polymeric film further comprises at leastone hole and wherein said hole is positioned over at least one of saidmicro-pores.
 6. The device of claim 5, wherein said device furthercomprises a pressure source or electrolytic expulsion source configuredproximal to said at least one hole.
 7. (canceled)
 8. The device of claim1, wherein said micro-pores further comprise at least one biologicalcell.
 9. (canceled)
 10. The device of claim 1, wherein said devicecomprises a micro-pore testbed array, said array comprising a pluralityof longitudinally fused fibers reversibly bonded to a single gaspermeable solid substrate, wherein said solid substrate is degassed andwherein said solid substrate is attached to at least one bindingpartner.
 11. (canceled)
 12. The device of claim 10, comprising betweenapproximately 300 to 11,500,000 of said fused fibers, per cm² of thearray.
 13. The device of claim 1, wherein said solid substrate comprisesa gas permeable material.
 14. The device of claim 10, wherein said gaspermeable material comprises poly(dimethylsiloxane) (PDMS).
 15. Thedevice of claim 10 comprising said plurality of longitudinally fusedfibers and a polymeric film, wherein said fused fibers are covered bysaid polymeric film, wherein said polymeric film further comprises atleast one hole and wherein said hole is positioned over at least one ofsaid fused fibers.
 16. The device of claim 15, wherein said devicefurther comprises a pressure source configured proximal to said at leastone hole. 17.-18. (canceled)
 19. A method for identifying asub-population of cells from a heterologous population of biologicalcells, the method comprising: a) providing: i) an array of micro-pores,wherein the internal diameter of micro-pores ranges betweenapproximately 1.0 micrometers and 500 micrometers; ii) said heterologouspopulation of cells; iii) at least one binding partner; b) contactingsaid array with said heterologous population of cells and said at leastone binding partner such that a sub-population comprising at least oneof said biological cells settles into at least one of said micro-poresof said array; c) incubating said array under conditions to promote thesecretion of molecules from said biological cells; and d) detectingdesired secreted molecules in at least one of said micro-pores of saidarray, thereby identifying said sub-population of cells.
 20. The methodof claim 19, wherein the providing step comprises providing an array ofmicro-pores not being coated with at least one binding partner. 21-45.(canceled)